Antiseptic compositions and uses thereof

ABSTRACT

Antiseptic compositions and kits containing a source of silver ions and menthol, which act in synergy such that a concentration of silver ions in the composition is substantially reduced, are disclosed. Further disclosed are uses of the antiseptic compositions as disinfectants and in the treatment of wounds.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.12/703,853 filed on Feb. 11, 2010, which claims the benefit of priorityof U.S. Provisional Patent Application No. 61/151,539 filed Feb. 11,2009. The contents of the above applications are all incorporated hereinby reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates toantiseptic compositions and uses thereof and, more particularly, but notexclusively, to antiseptic compositions which can be efficiently used indisinfecting surfaces such as bodily surfaces, and thus can beefficiently used, for example, in the treatment of infection-associatedmedical conditions such as acute and chronic wounds, burns, and surgicalwounds.

Chronic wounds are characterized by an impaired healing process, aprolonged inflammatory stage, re-epithelization failure and defectiveextra-cellular matrix re-modeling. Cells accumulated within these woundsare senescent and their response to exogenous growth factors is reduced.The end result of the impaired healing process of the chronic wound istissue breakdown, local necrosis and infection.

Management of wound bed is a multistep process necessary to achieveultimate wound closure. Debridement, infection control, and woundclosure are fundamental stages in the healing of chronic wounds.

In 2005, hospital acquired infections was ranked as the fourth-leadingcause of death in the USA. It has been reported that approximately 10million patients with traumatic wounds are treated in US emergencydepartments annually. Chronic wounds affect 1-3% of the entirepopulation and include primarily venous leg ulcers, diabetic foot ulcersand pressure ulcers. Venous ulcers caused by chronic venousinsufficiency are the most frequent (˜70%) among the non-healing chronicwounds.

The number of emergency cases in which the infection of a chronic woundleads to life-threatening complications is increasing along with thegrowth in the number of chronic wounds infected with microorganismsresistant to antibiotics. The presence of bacterial colonization is oneof the most crucial factors in the pathogenesis of most chronic wounds,resulting in high bacterial counts in wound tissue and in inflammatoryhost response.

In infected chronic wounds, wound healing is delayed or even abrogatedwhen infected with heavy bacterial burden. The reduction of bacterialload is crucial and should be carried out prior to the healing process.In order to optimize the wound healing process, treatment of underlyingfactors such as malnutrition and ischemia along with decreasingbacterial load is often necessary.

Infection of chronic wounds delays healing and usually causesdeterioration in the condition of the chronic wound bed, which can alsoresult in complete breakdown of the wound. Apart from the detrimentaleffect on wounds, infection can cause systemic effect, which in somecases can be fatal.

Bacterial contamination delays wound healing through several differentmechanisms e.g., persistent production of inflammatory mediatorsresulting in a prolonged inflammatory response that contributes to hostinjury and delays the healing process. In addition, bacteria competewith host cells for nutrients and oxygen essential for the wound healingprocess. Wound infection can also lead to tissue hypoxia, interrupt thedevelopment of granulation tissue, reduce the number of fibroblasts andcollagen production and damage re-epithelization. Addressing thequestion of infected wound cleansing is therefore a major objective ofwound care and plays a critical role in wound management.

A large fraction of foot ulcers results from complication of diabetesmellitus. These lesions frequently become infected and are oftenaccompanied by osteomyelitis. Most infections in these lesions are mildto moderate in severity and can be managed with appropriate wound careand oral antibiotic therapy. Some infections, however, penetrate to thefascia, muscle, joint and bone. In theses cases, the patients requirehospitalization, parenteral antibiotic therapy and surgical procedures.In some cases, foot infections in patients with diabetes can bedifficult to treat and therapeutic failure often leads to alower-extremity amputation. In addition, diabetic foot infections causedby methicillin-resistant S. aureus (MRSA) are associated with delayedhealing an in many cases with amputations.

Antiseptic agents are commonly used to prevent and treat woundinfections. Unlike antibiotics which act selectively on specificmicroorganisms, the antiseptic agents have multiple targets and abroader spectrum of activity. Most of the practiced antiseptic agentshave not been shown to clearly impede healing.

Among the commercially available antiseptic agents the most commonlyused in clinical practice are iodine, chlorhexidine, alcohol, acetate,hydrogen peroxide, boric acid, silver nitrate, silver sulfadiazine andsodium hypochlorite. Their efficacy, however, seems to be limited inview of recently reported outbreaks associated with contaminatedantiseptics [Weber et al. 2007, Antimicrob Agent Chemother 51:4217-4224].

Silver compounds have been widely used as wound antiseptics to counterbacterial infections in chronic and acute wounds including burns[Burrell 2003, Ostomy Wound Manag 49:19-24; Ovington 2004, Ostomy WoundManag 50:1S-15]. Silver is known to block the growth of gram-negativeand gram-positive bacteria. Ionic silver kills microorganisms but it isnon-cytotoxic to proliferating granulation tissue. Silver formulationsappear to increase the rate and degree of microbial killing, decreaseexudate formation and recently reported to affect bacterial biofilms.

The most commonly used silver compounds are silver sulfadiazine (SSD)and silver nitrate (AgNO₃). Other commonly employed topical silvercontaining agents are nanocrystalline silver particles. Silversulfadiazine (SSD) is usually used as a treatment for the prevention ofinfection in patients with burn wounds [Klasen 2000, Burns 26: 131-138].Nanocrystalline silver releasing systems, e.g., Acticoat®[Tredget et al.1998, J. Burn Care Rehabil. 19: 531-537] were developed in order toprolong its efficacy. Patients with infected chronic pressure ulcerswere subjected to SSD and effective decreasing of the bacterial loadwere observed in all ulcers. SSD 1% cream significantly reduced venousulcer, positively affecting wound cleansing and granulation tissueformation.

Silver nitrate was used first for the treatment of chronic wounds andulcers [Klasen 2000, Burns 26: 131-138]. It was found to be effectiveagainst a wide range of bacterial flora especially against gram-negativebacteria. Nanocrystalline silver was incorporated later into wounddressings as a sustained release formulation for the treatment of acuteand chronic wounds including burns [Voight et al. 2001, Wounds 13:B11-B21; Sondi and Salopek-Sondi 2004, J Colloid Interf Sci 275:177-182; Parsons et al. 2005, Wounds, 17: 222-232].

Silver containing formulations for topical wound treatment are mostlybased on silver containing wound dressing. The delivery and penetrationof silver ions into the infected wound bed by such dressings is oftenlimited by chemical reactions e.g. with chloride ions resulting inprecipitation of inactive silver chloride, reducing the effective amountof antimicrobial silver ions reaching the targeted infected areas. Otherdrawbacks include silver affected coloration and local irritation.

In parallel with the common and excessive use of silver based topicaltreatments for wound management an increase in the number of reports onbacterial silver resistance was noticed [Silver 2003, FEMS MicrobiologyReviews 27:341-353; Chopra 2007, J Antimicrobial Chemotherapy 59:587-590].

Menthol is a natural monoterpene of plant origin, frequently used indermatology as part of antipruritic, antiseptic, analgesic and/orcooling formulations. The molecular mechanisms underlying mentholcontribution to these activities—and in particular to its antibacterialactivity—were recently described [Patel, 2007, J Am Acad Dermatol 57:873-878; Evrand 2001, Pharma Resear 18:943-949; Trombetta et al. 2005,Antimicrob Agent Chemother 49: 2474-2478; Schelz et al. 2006, Fiterapia77: 279-285; Cristani et al. 2007, J Agri Food Chem 55: 6300-6308].

Hypertonic solutions are expected to add benefits to wound bedmanagement by reduction of microbial load, enhanced exudate removal andimpact on fluid circulation. Hypertonic saline solutions have beenproven to be a very useful tool in neurosurgical practice.

Art of relevance include U.S. Pat. Nos. 5,643,589 and 5,562,643

Menthol has been described in the art as an antipruritic agent,antiseptic agent, analgesic and cooling agent, which can be used also indermatological applications. Compositions comprising silver ions andmenthol have also been described.

For example, The Merck Manual suggests combining menthol and silver indermatological compositions in the chapter “Principles of TopicalDermatologic Therapy”(http://www.merck.com/mmpe/print/sec10/ch110/ch110a.html). Silver isdefined as an antiseptic agent and menthol is defined as ananti-pruriritic agent (anti-itching agent) therein.

Additional relevant art include International Patent Application No.PCT/IL2007/000015, U.S. Pat. No. 6,551,608, U.S. patent application Ser.No. 11/783,668 (Publication No. 20070255193) and U.S. patent applicationSer. No. 10/535,961 (Publication No. 20060105000),

Antiseptic compositions comprising silver ions have also been disclosedin U.S. Pat. No. 5,607,683 and U.S. Pat. No. 6,093,414.

A continuous Streaming of Therapeutic solution (CST) is a new modalityof chronic wound management developed by the present inventors, as wellas others, and involves the continuous streaming of fresh doses oftherapeutic solutions into the controlled wound environment. Streamingover chronic wounds allows for aseptic confinement of the wound,negative pressure therapy (pump-free), moist conditions, continuouscleansing and overall management of the wound bed. Such ContinuousStreaming therapy has been disclosed, for example, in U.S. Pat. No.7,364,565, to some of the present inventors.

SUMMARY OF THE INVENTION

In a search for an antiseptic composition with improved performance, thepresent inventors have surprisingly uncovered that a combination ofsilver ions and menthol exhibits a synergistic effect.

It has been further uncovered that this activity is retained in thepresence of a hyperosmotic agent.

Thus, it is disclosed herein that silver ions-based antisepticcompositions exhibit a synergistic effect when combined with lowconcentration of menthol, in isotonic or hypertonic medium, wherein thesilver ions are present at lower concentrations than are typically beingused for antiseptic purposes. Such compositions are capable of exertingantimicrobial activity against a broad spectrum of pathogenicmicroorganisms while allowing pain relief, cooling and wound healing,without being associated with adverse side effects such as colorationand irritation, which are frequently observed for currently availablesilver ions-containing solutions.

It has further been demonstrated that antiseptic compositions comprisingsilver ions or complexes thereof at a concentration ranging from 0.005%w/v to 0.5% w/v in combination with menthol at a concentration ranging0.05% w/v to 0.5% w/v and a hyperosmotic agent such as glycerol, areclear solutions devoid of precipitates.

Accordingly, acute and chronic wounds such as diabetic ulcers can beefficiently treated with the antiseptic compositions described herein.Moreover, higher rates of wound healing can be achieved when theantiseptic compositions described herein are applied over and throughthe wound in continuous flow so that the concentrations of theantimicrobial agent and the hyperosmotic agent in contact with the woundare kept constant.

It has further been demonstrated that such antiseptic compositions mayact as preservatives, by preventing or reducing microbial growth innon-sterile conditions.

It has further been demonstrated that such antiseptic compositionsexhibit effective fungicidal activity, which outperforms commerciallyavailable antifungal products. It has further been demonstrated that acombined, sequential treatment of an antiseptic composition as describedherein and a polyhexadine-based commercial antiseptic product is highlyeffective in inhibiting growth of various fungal strains, particularlywhen the silver antiseptic composition is used first.

According to an aspect of some embodiments of the invention there isprovided an antiseptic composition comprising, as active ingredients,menthol and a source of silver ions, and a pharmaceutically acceptablecarrier, wherein a concentration of the silver ions in the compositionis lower than 6 mM.

In some embodiments, the menthol and the silver ions act in synergy.

In some embodiments, a concentration of the menthol ranges from 0.3 mMto 32 mM.

In some embodiments, the concentration of the menthol ranges from 0.6 mMto 6.4 mM.

In some embodiments, a concentration of the silver ions ranges from 0.05mM to 6 mM.

In some embodiments, a concentration of the silver ions ranges from 0.25mM to 0.6 mM.

In some embodiments, a concentration of the silver ions ranges from 0.25mM to 0.6 mM, and a concentration of the menthol ranges from 0.6 mM to6.4 mM.

In some embodiments, a concentration of the silver ions is 0.29 mM, anda concentration of the menthol is 6.4 mM.

In some embodiments, a concentration of the silver ions is 0.29 mM, anda concentration of the menthol is 3.2 mM.

In some embodiments, a concentration of the silver ions is 0.29 mM, anda concentration of the menthol is 0.64 mM.

In some embodiments, a concentration of the silver ions is 0.44 mM, anda concentration of the menthol is 3.2 mM.

In some embodiments, a concentration of the silver ions is 0.6 mM, and aconcentration of the menthol is 3.2 mM.

In some embodiments, a concentration of the silver ions is 0.6 mM, and aconcentration of the menthol is 0.64 mM.

In some embodiments, the source of silver ions is selected from thegroup consisting of silver nitrate, silver sulfadiazine, anaminoalcohol-silver ion complex, an amino acid-silver ion complex and apolymer-silver ion complex.

In some embodiments, the polymer-silver ion complex ispolyvinylpyrrolidone (PVP)-silver ion complex.

In some embodiments, the antiseptic composition further comprises ahyperosmotic agent.

In some embodiments, the hyperosmotic agent is selected from the groupconsisting of glycerol, polyethylene glycol, a polysaccharide, mannitol,and a combination thereof.

In some embodiments, the hyperosmotic agent is glycerol.

In some embodiments, a concentration of the glycerol ranges from 3% v/vto 15% v/v, based on the total volume of the antiseptic composition.

In some embodiments, a concentration of the glycerol is 10% v/v, basedon the total volume of the antiseptic composition.

In some embodiments, the hyperosmotic agent is polyethylene glycol(PEG).

In some embodiments, a concentration of the PEG ranges from 8% v/v to16% v/v based on the total volume of the antiseptic composition.

In some embodiments, the antiseptic composition is further comprising asolubilizing agent.

In some embodiments, the solubilizing agent is TWEEN 20.

In some embodiments, the pharmaceutically acceptable carrier is anaqueous solution.

In some embodiments, the antiseptic composition is formulated as atopical dosage form.

In some embodiments, the topical dosage form is selected from the groupconsisting of a cream, a spray, a gauze, a wipe, a sponge, non-wovenfabrics, a cotton fabrics, a foam, a solution, a lotion, an ointment, apaste and a gel.

According to an aspect of embodiments of the invention there is providedan antiseptic kit comprising a packaging material and the antisepticcomposition as described herein being packaged in the packagingmaterial.

In some embodiments, the antiseptic kit is being identified in print, inor on the packaging material, for use in disinfecting a surface.

In some embodiments, the surface is a bodily surface.

In some embodiments, the antiseptic kit is identified in print, in or onthe packaging material, for use in the treatment of a wound.

In some embodiments, the wound is selected from the group consisting ofan acute wound, a chronic wound, a burn and a surgical wound.

In some embodiments, the chronic wound is selected from the groupconsisting of a diabetic ulcer, a venous ulcer and a pressure ulcer.

In some embodiments, at least one of the source of silver ions, thementhol and the pharmaceutically acceptable carrier is individuallypackaged within the packaging material.

In some embodiments, each of the source of silver ions, the menthol andthe pharmaceutically acceptable carrier is individually packaged withinthe packaging material.

In some embodiments, the source of silver ions, the menthol and thepharmaceutically acceptable carrier are packaged together within thepackaging material.

According to an aspect of embodiments of the invention there is provideda method of disinfecting a surface, the method comprising applying aneffective amount of the antiseptic composition as described herein ontothe surface, thereby disinfecting the surface.

In some embodiments, the surface is a bodily surface, the method beingfor disinfecting the bodily surface of a subject in need thereof.

In some embodiments, the method comprises topically applying theantiseptic composition onto the bodily surface.

In some embodiments, the bodily surface is a skin tissue.

In some embodiments, the method is being for treating an infection insaid bodily surface.

In some embodiments, the infection is caused by a pathogenicmicroorganism selected from the group consisting of bacteria, yeast andfungi.

According to an aspect of embodiments of the invention there is provideda method of treating a wound, as described herein, in a subject in needthereof, the method comprising applying an effective amount of theantiseptic composition as described herein to the wound area, therebytreating the wound.

In some embodiments, the method comprises topically applying theantiseptic composition onto the wound area.

In some embodiments, topically applying the antiseptic composition isperformed by streaming a flow of the antiseptic composition over andthrough the wound area.

In some embodiments, the flow is induced by gravity from at least onereservoir that comprises the antiseptic composition.

In some embodiments, the flow is induced by a pump being in fluidcommunication with at least one reservoir that comprises the antisepticcomposition.

According to an aspect of some embodiments of the invention there isprovided a use of the antiseptic composition described herein in themanufacture of a product for disinfecting a surface.

In some embodiments, the product is a medicament for disinfecting abodily surface.

In some embodiments, the medicament is for treating an infection in saidbodily surface.

In some embodiments, the infection is caused by a pathogenicmicroorganism selected from the group consisting of bacteria, yeast andfungi.

According to an aspect of some embodiments of the invention there isprovided a use of the antiseptic composition described herein in themanufacture of a medicament for treating a wound, as described herein.

According to an aspect of some embodiments of the invention there isprovided a process of preparing the antiseptic composition as describedherein, the process comprising admixing the source of silver ions, thementhol and the pharmaceutically acceptable carrier, thereby obtainingthe antiseptic composition.

In some embodiments, the method further comprises admixing ahyperosmotic agent with the composition.

In some embodiments, the method further comprises admixing asolubilizing agent with the composition.

In some embodiments, the solubilizing agent is admixed with the menthol,prior to admixing the menthol with the source of silver ions and thecarrier.

In some embodiments, the solubilizing agent is TWEEN 20.

According to an aspect of some embodiments of the invention there isprovided a method of reducing a concentration of silver ions in anantiseptic composition which comprises silver ions, the methodcomprising admixing with a source of the silver ions a synergisticallyeffective amount of menthol, thereby reducing the concentration of thesilver ions in the antiseptic composition.

In some embodiments, the concentration of the silver ions is reduced byat least 2-folds.

In some embodiments, the concentration of the silver ions is reduced byat least 10-folds.

According to an aspect of some embodiments of the invention there isprovided a method of increasing an antiseptic activity of an antisepticcomposition which comprises a source of silver ions at a concentrationlower than 6 mM, the method comprising admixing with the composition asynergistically effective amount of menthol.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the images makes apparent to those skilled in theart how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 presents an image generally illustrating the appearance of afungal culture following 24 days incubation after 30 minutes exposure toa tested solution and subsequent washing, and presenting no growth(Fungicidal effect, denoted as “F”), minimal poor growth (Inhibitoryeffect, denoted as “IN”) or full growth (No Effect, denoted “NE”).

FIGS. 2, 3 and 4 present images illustrating the appearance of theexposed fungal culture, following 3, 8, 11 and 16 days incubation after30 minutes exposure to each of the tested solutions and subsequentwashing. Tested solutions included: a composition containing silver ionsand menthol as described in Example 5 hereinabove (denoted “S”),PRONTOSAN® (denoted “P”), alternating subsequent exposures to acomposition containing silver ions and menthol (S) and PRONTOSAN® (P),(denoted “S+P” and “P+S”), MICROCYN® (denoted “M”) and ANACEPT® (denoted“A”) and their dilutions (1:2; 1:4; 1:8). Tested fungal strains were aclinical strain of Trichophyton rubrum (FIG. 2); Trichophyton rubrumNCPF 118 commercial strain (FIG. 3); and a clinical strain ofMicrosporum canis (FIG. 4).

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates toantiseptic compositions and uses thereof and, more particularly, but notexclusively, to antiseptic compositions which can be efficiently used indisinfecting surfaces such as bodily surfaces, and thus can beefficiently used, for example, in the treatment of infection-associatedmedical conditions such as acute and chronic wounds, burns, and surgicalwounds, and/or as preservatives.

The antiseptic compositions described herein comprise a source of silverions and menthol, whereby the silver ions and the menthol act insynergy, thus allowing use of relatively low concentrations of each. Theantiseptic compositions described herein are therefore readily preparedas solutions (as an example), with no precipitation of the activeingredients, and are devoid of the adverse side effects associated withcurrently available silver ions-based antiseptics while exhibiting highand broad therapeutic efficacy.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

The present inventors have recognized that one of the potentialsolutions to the development of microbial resistance to silver is theuse of a combination of a source of silver ions, and anotherantibacterial agent which, together with the silver ions, exhibits anantibacterial (or antimicrobial) synergistic effect. Such a combinationenables substantial reduction in silver input, and thus also eliminatesside effects such as wound coloration and irritation caused by silverions. Such a combination has, however, to be based on compatibility ofall ingredients used with silver ions, so as to avoid potentialprecipitation of silver. Provided that suggested combinations arecompatible, tests are readily available for feasibility demonstration ofthe synergistic antimicrobial effect (see, for example, Mackay et al.2000 Int J Antimicrob Agents 15:125-129; and Peter et al. 2006, JAntimic Chemother 57: 573-576).

While many antibacterial, antimicrobial and/or antiseptic agents areknown, it is difficult to predict which of these agents, when combinedtogether, would act in synergy.

The term “antiseptic” as used herein describes the capability of anagent or a composition to effect reduction in microbial load, and/or toeffect prevention of development of microbial load, by topicalapplication of the agent or composition. An antiseptic composition oragent is thus capable of preventing or arresting the growth or action ofmicroorganisms (such as, for example, bacteria, yeast and fungi) eitherby inhibiting their activity and/or growth or by destroying (killing)the microorganisms.

The term “antiseptic” is commonly used in the art to describepreparations for topical application to the infected living tissue or toany other infected surface, or a surface at risk of being infected, asis detailed hereinbelow.

Antiseptic compositions and agents are also referred to herein asantimicrobial compositions or agents. The term “antimicrobial”, as usedherein, describes a composition or agent capable of preventing orarresting the growth and/or action of microorganisms either byinhibiting the activity of the microorganisms, by inhibiting the growthof the microorganisms or by killing the microorganisms.

The terms “synergy”, “synergism”, and any grammatical diversion thereof,as used herein, describe a cooperative action encountered incombinations of two or more biologically active compounds in which thecombined effect exhibited by the two compounds when used togetherexceeds the sum of the effect of each of the compounds when used alone.“Synergy” is therefore often determined when a value representing aneffect of a combination of two active agents is greater than the sum ofthe same values obtained for each of these agents when acting alone.

A synergy between two antiseptic agents may be determined by methodswell known in the art.

As presented in the Examples section that follows, it has beensurprisingly uncovered that silver ions and menthol act in synergy.Synergy was determined by measuring the antiseptic activity of mentholalone and silver ions alone, against various bacteria, compared to theiractivity when administered together. The two agents are considered toact in synergy if the observed antiseptic activity of the agents, whenadministered together, is in excess of the cumulative antisepticactivity expected by combining the observed activity of the agents whenadministered alone.

In addition, synergy was demonstrated, and thus determined, whenpharmacokinetic parameters of the antiseptic activity, exhibited whenthe agents are administered together, are superior to the samepharmacokinetic parameters of the antiseptic activity of the agents wheneach agent is administered alone. An exemplary pharmacokinetic parameteris the time length required for achieving a certain level of anantiseptic effect. Thus, the two agents are considered to act in synergyif the time length required for achieving this level of antisepticeffect is reduced when the agents are administered together, as comparedto this time length when the agents are administered each alone.

It is noted in this regard that in pharmacokinetic parameters, usuallythere is no significance for the sum of values, and hence, anyimprovement in a pharmacokinetic parameter upon co-administration of twoagents, relative to administration of each compound alone, is consideredto reflect synergy.

Thus, it has been shown, for example, that synergistic antisepticactivity was observed for hyperosmotic solutions containing the silverion source AgNO₃ and menthol at concentrations of 0.005% w/v %+0.1% w/v,0.005% w/v+0.05% w/v, 0.005% w/v+0.01% w/v, 0.0075% w/v+0.05% w/v, 0.01%w/v+0.05% w/v and 0.01% w/v+0.01% w/v, respectively, when tested againstE. coli ATCC47076 strain (see, Table 1 hereinbelow). These valuescorrespond to a molar concentration for silver ions and menthol of 0.29mM+6.4 mM, 0.29 mM+3.2 mM, 0.29 mM+0.64 mM, 0.44 mM+3.2 mM, 0.6 mM+3.2mM and 0.6 mM+0.64 mM, respectively. Specifically, at theseconcentrations, the cumulative antiseptic activity of menthol and ofsilver ions when each administered alone was lower than the antisepticactivity observed when the agents were administered together.

It has been also shown that silver ions and menthol act in synergyagainst various types of bacteria, including Klebsiella pneumoniae, aMethicillin Resistant clinical strain of Staphylococcus aureus (MRSA), aclinical strain of Staphylococcus epidermis, a clinical strain ofExtended Spectrum B-lactamase producing Escherichia coli, a clinicalstrain of Multi Drug Resistant Acinetobacter baumannii, and a clinicalstrain of Multi Drug Resistant Pseudomonas aeruginosa (see, Table 2hereinbelow). In these studies, the synergistic effect of the silverions and menthol was demonstrated by the shorter time length measureduntil a certain level of antiseptic activity was observed as compared tothe time length until the same level of antiseptic activity was observedwhen each agent was administered alone.

It has been further shown that solutions containing silver ion sourceand menthol are clear solutions, devoid of precipitates.

It has further been demonstrated that the minimum inhibitoryconcentration of such a composition is obtained upon 1:4 dilution of thecomposition, and that the minimum bactericidal concentration of such acomposition is obtained upon 1:2 dilution of the composition (see, Table3 in the Examples section the follows).

Accordingly, an antiseptic composition as described herein can bebeneficially used in any of the methods described herein, while beingdiluted by at least 1:2, and optionally by 1:2.1, 1:2.2, 1:2.3, 1:2.4,1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3, 1:3.1, 1:3.2, 1:3.3, 1:3.5,1:3.5, 1:3.6, 1:3.7, 1:3.8, 1:3.9, 1:4, and even upon higher dilutions(e.g., 1:5, 1:6, 1:7, 1:8, 1:9 and 1:10).

It has been further shown that upon 1:10 dilution, an exemplarycomposition comprising silver ions and menthol prevented growth ofvarious bacterial and fungal strains in a non-sterile sample (see,Example 5 in the Examples section that follows).

The beneficial therapeutic effect of the antiseptic compositionsdescribed herein was further demonstrated in comparison with thecommercially available products PRONTOSAN®, MICROCYN® and ANASEPT® (see,Example 6 in the Examples section that follows, and FIGS. 2-4). Theantiseptic compositions described herein were shown to exhibitfungicidal activity against clinical fungal strains such as Trichophytonrubrum and Microsporum canis, which outperformed some of thecommercially available products, and was found to be effectivelycombined with PRONTOSAN®.

The synergistic effect observed for the silver ions and mentholcombination therefore enables to provide antiseptic compositions withreduced concentration of silver ions, along with taking advantage ofother beneficial properties of menthol, such as, for example, itsantipruritic effect, its coolant effect, etc.

These findings demonstrate that a composition comprising silver ions andmenthol may serve as potent antiseptic composition, for disinfectingsurfaces such bodily surfaces, and hence for the treatment or preventionof infection-associated conditions such as wounds, as detailedhereinunder.

These findings further demonstrate that a composition comprising silverions and menthol may serve as potent antiseptic composition, fordisinfecting surfaces of, for example, medical devices and storagecontainers, and for preventing development of microbial load on suchsurfaces.

Thus, according to an aspect of some embodiments of the invention thereis provided an antiseptic composition comprising, as active ingredients,menthol and a source of silver ions, and a pharmaceutically acceptablecarrier, wherein the concentration of the silver ions in the compositionis lower than 6 mM.

According to some embodiments, the menthol and the silver ions act insynergy, as defined hereinabove.

Menthol, (5-methyl-2-propan-2-yl-cyclohexan-1-ol; CAS No. 89-78-1,MW=156 grams/mol) is a natural monoterpene of plant origin, frequentlyused in dermatology as part of antipruritic, antiseptic, analgesic andcooling formulations. Menthol is a waxy, crystalline substance, clear orwhite in color, which is solid at room temperature and melts slightlyabove it. Menthol has three asymmetric centers and exists as eightstereoisomers: (+)-menthol, (+)-isomenthol, (+)-neomenthol,(+)-neoisomenthol, (+)-menthol, (+)-isomenthol, (+)-neomenthol,(+)-neoisomenthol, all being encompassed by the term “menthol” as usedherein.

The most common form of menthol occurring in nature is the (−)-mentholstereoisomer, which is assigned the (1R,2S,5R) configuration and has thefollowing structure:

The phrase “a source of silver ions”, as used herein, describes achemical moiety which generates silver ions when present in anappropriate medium. The silver ions can be generated once the compoundis mixed with the carrier of the composition as described herein (as inthe case of, for example, water-soluble silver salts in an aqueouscarrier). Alternatively, the silver ions can be generated upondecomposition of a chemical complex that contains the silver ions, adecomposition that occurs once the complex is placed in the carrier ofthe composition. In such chemical complexes, the silver ions aretypically engaged in coordinative interactions, while in silver salts,the silver ions are engaged in ionic interactions.

In some embodiments, the pharmaceutically acceptable carrier is anaqueous solution. In these embodiments, the source of silver ions ispreferably a water-soluble silver salt such as, for example, silvernitrate, silver acetate and the partially water-soluble silversulfadiazine.

Non-limiting examples of sources of silver ions that are suitable foruse in the context of the present embodiments include silver nitrate,silver sulfadiazine, and silver ion complexes such as anaminoalcohol-silver ion complex, an amino acid-silver ion complex and apolymer-silver ion complex.

The phrase “aminoalcohol-silver ion complex” describes a complex ofamino alcohol (i.e. a molecule which contains both an amine functionalgroup and an alcohol functional group) with silver ions. Non-limitingexamples of such complexes include aminoethanol, amino propyl alcohol,2-amino-2-(hydroxymethyl)-1,3-propanediol, and aminobutyl alcohol.

In some embodiments, the concentration of the aminoalcohol component isabout 50 mM.

The phrase “amino acid-silver ion complex” describes a complex of anamino acid (i.e., a molecule which contains both an amine functionalgroup and a carboxylic acid functional group) with silver ions. In someembodiments, the amino acid is selected from the group of naturallyoccurring amino acids, non-natural amino acids and amino acid analogs.Non-limiting examples of such complexes include a histidine-silver(I)complex, a serine-silver complex and a lysine-silver complex.

The phrase “polymer-silver ion complex” describes a complex of a polymerwith silver ions. The polymer in such a complex should have one or morefunctional groups (e.g., hydroxy, amine, oxo (═O), carboxylate, amideand the like) that coordinatively interact with the silver ions. Thepolymer may be synthetic, naturally-occurring or semi-synthetic polymerand is preferably biocompatible. Non-limiting examples of suchpolymer-silver ion complexes include polyvinylpyrrolidone (PVP)-silverion complex, polyacrylamide-silver ion complex and polylysine-silver ioncomplex.

According to some embodiments, the polymer-silver ion complex ispolyvinylpyrrolidone (PVP)-silver ion complex.

Polyvinylpyrrolidone is a water-soluble polymer with an excellent safetyprofile and a wide variety of applications in medicine, pharmacology,cosmetics and industrial production. Polyvinylpyrrolidone readily formsfilms and is used as a binder in many pharmaceutical tablets.

In some embodiments, where the silver ion source is apolyvinylpyrrolidone-silver ion complex, the polymer's concentration inthe antiseptic composition ranges from 0.2% w/v to 10% w/v, based on thetotal volume of the antiseptic composition.

As used herein, “% w/v” describes a weight percentage of a component oran agent in the total volume of the composition. Thus, for example, 1%w/v describes 1 mg of a component or an agent in 100 ml of thecomposition.

The “% w/v” units, while expressing a mass present in a certain volume,can also be expressed as molarity, namely, moles per liter (M).

“% v/v” units describe a volume percentage of a component or an agent inthe total volume of the composition.

As discussed hereinabove, the surprising findings that menthol and thesilver ions act in synergy, allow utilizing an antiseptic compositionthat contains reduced concentrations of each agent, as compared to theconcentration of each agent that is typically utilized for exerting anantiseptic activity, while still obtaining the desired antisepticactivity.

For example, silver nitrate-based antiseptic solutions at aconcentration of 0.5% w/v are typically used to treat severe burns[Sweetman S C (ed) Martindale: The complete drug reference 35, London:Pharmaceutical press, 2007].

As further discussed hereinabove, such a concentration is relativelyhigh and is associated with adverse effects such as wound coloration andirritation.

A concentration of 0.5% w/v silver nitrate corresponds to about 30 mM.

The concentration of menthol in currently known compositions for topicaladministration ranges between 0.25 w/v %, applied to the nasal mucousmembrane for high fever and catarrh (a thick exudate of mucus) and up to20% w/v for treatment of neuralgia, sciatica and lumbago.

The above concentration range of menthol corresponds to from about 15 mMto about 1.3 M.

It is noted herein that while the antiseptic properties of menthol areknown, an indication of a recommended concentration of menthol inantiseptic composition could not be found in the art.

As exemplified in the Examples section that follows, silver nitratesolutions of concentrations lower than currently acceptable inantiseptic compositions (i.e., from 0.005% w/v to 0.01% w/v,corresponding to a range of from about 0.29 mM to about 0.6 mM) havelimited antiseptic activity unless combined with menthol (see, Table 1).The addition of menthol, at a concentration of from 0.01 w/v % to 0.1w/v %, corresponding to a concentration of from about 0.6 mM to about 6mM, leads to a substantial enhancement of the antiseptic activity.

As further exemplified in the Examples section that follows, the limitedantiseptic activity of silver nitrate solutions at the above-indicatedlower concentrations is further demonstrated by the time length requiredfor exhibiting a bactericidal activity (see, Table 2). The addition oflow concentrations of menthol leads to substantial reduction of thistime length.

Without being bound to any particular theory, it is hypothesized thatthe synergistic, antiseptic activity observed when silver ions areadministered together with menthol results, in addition to theantiseptic activity exerted by menthol, also from the menthol ability tofunction as a penetration enhancer, which enhances the rate ofpenetration and the amount of silver ions penetrating into an infectedarea and/or microbial cells.

Thus, according to some embodiments of the invention, the concentrationof the menthol in the antiseptic composition described herein rangesfrom about 0.3 mM to about 35 mM. This concentration range correspondsto a % w/v menthol concentration in a range of from 0.005% w/v to 0.5%w/v, based on the total volume of the antiseptic composition.

In some embodiments, the concentration of menthol ranges from about 0.6mM to about 6.4 mM. This molar concentration range corresponds to a %w/v menthol concentration in a range of from 0.01% w/v to 0.1% w/v,based on the total volume of the antiseptic composition.

In some embodiments, the concentration of the source of silver ions isselected so as to provide a concentration of the silver ions in thecomposition which ranges from about 0.05 mM to about 6 mM.

Thus, in some embodiments the concentration of the source of silver ionsis such that the concentration of silver ions in the composition is, forexample, 0.05 mM, 0.06, mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM, 0.2 mM,0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, 0.8 mM, 0.9 mM, 1 mM, 1.5 mM, 2mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM or 6 mM. Any othervalue of silver ions concentration between 0.05 mM and 6 mM is alsocontemplated.

In some embodiments, the silver ion source is AgNO₃ and theconcentration of AgNO₃ ranges from 0.001% w/v to 0.5% w/v, based on thetotal volume of the antiseptic composition. In some embodiments, theconcentration range of AgNO₃ is from 0.005% w/v to 0.01% w/v, based onthe total volume of the antiseptic composition. In some embodiments, theconcentration of the AgNO₃ ranges from 0.001% w/v to 0.5% w/v, and theconcentration of the menthol ranges from 0.005% w/v to 0.5% w/v, basedon the total volume of the antiseptic composition. In some embodiments,the concentration of the AgNO₃ ranges from 0.001% w/v to 0.5% w/v, andthe concentration of the menthol ranges from 0.005% w/v to 0.5% w/v,based on the total volume of the antiseptic composition.

A concentration of the source of silver ions that provides such aconcentration of silver ions can be readily determined based on thesolubility and/or degree of decomposition of the source of silver ionsin the selected carrier, its molecular weight and the number of silverions that are generated from each molecule of the silver ion source.

In some embodiments, the concentration of the silver ions ranges from0.29 mM to 0.6 mM.

The concentration of silver ions present in the antiseptic compositiondescribed herein can therefore be, for example, 0.06 mM (0.001% w/vAgNO₃), 0.29 mM (0.005% w/v AgNO₃), 0.6 mM (0.01% w/v AgNO₃), 2.9 mM(0.05% w/v AgNO₃) or 5.9 mM (0.1% w/v AgNO₃), all being substantiallylower than the commonly used concentration of silver ions in currentlyavailable antiseptic compositions.

In some embodiments of the invention, the concentration of the silverions ranges from 0.05 mM to 6 mM, and the concentration of the mentholranges from 0.29 mM to 32 mM.

In some embodiments, the concentration of the silver ions ranges from0.29 mM to 0.6 mM, and the concentration of the menthol ranges from 0.6mM to 6.4 mM.

In one embodiment, the concentration of the silver ions is 0.29 mM, andthe concentration of the menthol is 6.4 mM,

In another embodiment, the concentration of the silver ions is 0.29 mM,and the concentration of the menthol is 3.2 mM.

In another embodiment, the concentration of the silver ions is 0.29 mM,and the concentration of the menthol is 0.64 mM.

In another embodiment, the concentration of the silver ions is 0.4 mM,and the concentration of the menthol is 3.2 mM.

In another embodiment, the concentration of the silver ions is 0.6 mM,and the concentration of the menthol is 3.2 mM.

In another embodiment, the concentration of the silver ions is 0.6 mM,and the concentration of the menthol is 0.64 mM.

In another embodiment, when the source of silver ions is AgNO₃, theconcentration of the AgNO₃ is 0.01% w/v, and the concentration of thementhol is 0.05% w/v, based on the total volume of the antisepticcomposition.

In another embodiment, the concentration of the AgNO₃ is 0.01% w/v, andthe concentration of the menthol is 0.01% w/v, based on the total volumeof the antiseptic composition.

In another embodiment, the concentration of the AgNO₃ is 0.0075% w/v,and the concentration of the menthol is 0.05% w/v, based on the totalvolume of the antiseptic composition.

In another embodiment, the concentration of the AgNO₃ is 0.005% w/v, andthe concentration of the menthol is 0.1% w/v, based on the total volumeof the antiseptic composition.

In another embodiment, the concentration of the AgNO₃ is 0.005% w/v, andthe concentration of the menthol is 0.05% w/v, based on the total volumeof the antiseptic composition.

In another embodiment, the concentration of the AgNO₃ is 0.005% w/v, andthe concentration of the menthol is 0.01% w/v, based on the total volumeof the antiseptic composition.

In some embodiments of the invention, the antiseptic compositiondescribed herein further comprises a hyperosmotic agent.

The phrase “hyperosmotic agent”, as used herein, describes an agentwhich raises the osmotic pressure at a site where it is applied. Ahyperosmotic agent is intended to increase the osmotic pressure aroundmicroorganisms such as bacteria, so as to kill or inhibit the growth ofthe microorganism. A composition which comprises a hyperosmotic agenthas a higher osmotic pressure than isotonic fluid. Typically, such acomposition has an osmotic pressure higher than a physiologicalpressure.

Exemplary hyperosmotic agents that are suitable for use in the contextof embodiments of the invention include, but are not limited to,glycerol, polyethylene glycol (PEG), a polysaccharide, mannitol and anycombination thereof.

In some embodiments, the hyperosmotic agent is compatible with thesource of silver ions and the menthol, such that no precipitation iseffected when it is present in the antiseptic composition.

Accordingly, a concentration of the hyperosmotic agent is selectedcompatible with their solubility in the pharmaceutically acceptablecarrier utilized in the antiseptic composition, so as to provide acomposition with an osmotic pressure that is higher than that of anisotonic solution.

In some embodiments, the hyperosmotic agent is biocompatible.

As used herein, the term “biocompatible” describes an agent orcomposition that is non-toxic and non-immunogenic when applied to aleaving organ, cell or tissue.

In some embodiments, the hyperosmotic agent is glycerol.

Glycerol is a chemical compound also commonly called glycerin. It is acolorless, odorless, viscous liquid and having low toxicity, which iswidely used in pharmaceutical formulations.

Glycerol is preferably present within the antiseptic composition at aconcentration of 3% v/v to 15% v/v, based on the total volume of thecomposition.

As used herein and in the art, a concentration unit of “% v/v” describesthe volume percents of an agent or component of the total volume of thecomposition. Thus, for example, 1% v/v represents 1 ml of an agent orcomponent in a 100 ml composition.

In some embodiments, the concentration of glycerol is 10% v/v.

In some embodiments, the hyperosmotic agent is polyethylene glycol(PEG).

PEG is a flexible, non-toxic (biocompatible), water-soluble polymer.PEGs are commercially available over a wide range of molecular weights,ranging from 300 DA to 10,000 KDa. Due to its characteristics, PEG canbe used to create osmotic pressures.

Exemplary PEGs that are suitable for use in this context of the presentembodiments include, but are not limited to, commercially available lowmolecular weight PEGs such as PEG 200 (Da), PEG 300 (Da) and PEG 400(Da). PEG is preferably present within the antiseptic composition at aconcentration of 8% to 16% v/v, based on the total volume of thecomposition.

As discussed hereinabove, it has been demonstrated that an antisepticcomposition as described herein forms a clear solution, devoid ofprecipitates. Such compositions are advantageous since they can bereadily applied on surfaces while utilizing any available technique fortopical application.

In some cases, solubilizing agents, or any other agents, are added tothe composition in order to facilitate the complete dissolution of itscomponents.

Thus, according to some embodiments of the invention, the compositionfurther comprises a solubilizing agent.

The term “solubilizing agent”, as used herein and in the art, describesa chemical agent which is capable of facilitating the dissolution ofinsoluble or poorly soluble components in a solution containing same.

A solubilizing agent, according to some embodiments of the invention,should be compatible with the source of silver ions used, so as to avoidprecipitation of non-soluble silver salts.

It is noted herein that most of the silver salts are insoluble or havelimited solubility in aqueous solutions, and hence any component that isadded to the antiseptic composition described herein should beconsidered by its effect on the silver solubility in the composition, inorder to maintain an effective concentration of soluble silver ions inthe composition.

Representative examples of solubilizing agents that are usable in thecontext of the present invention include, without limitation,complex-forming solubilizers such as cyclodextrin, polyvinylpyrrolidone,and micelle-forming solubilizers such as TWEENS and spans, e.g., TWEEN80 and TWEEN 20. Other solubilizing agents that are usable in thecontext of embodiments of the invention include, for example,polyoxyethylene sorbitan fatty acid ester, polyoxyethylene n-alkylethers, n-alkyl amine n-oxides, poloxamers, organic solvents,phospholipids and cyclodextrines.

The selection of the solubilizing agent is based on its being compatiblewith silver ions without causing precipitation of silver in the selectedcarrier.

As described in the Examples section that follows, menthol is used inthe composition described herein in combination with an appropriatesolubilizing agent, such as TWEEN 20 and the like. The solubilizingagent facilitates the formation of a stable and clear solutioncontaining the silver ion source and the menthol.

Thus, in some embodiments, the solubilizing agent is TWEEN 20. Accordingto some embodiments, the ratio between the menthol and TWEEN 20 is 1:10,e.g., 0.05% w/v menthol and 0.5% w/v TWEEN 20.

Without being bound to any particular theory, it is suggested that theaddition of TWEEN 20 to the antiseptic composition described herein mayfurther provide for enhanced activity of the composition due to thepenetration enhancing properties of TWEEN 20. Thus, it may be suggestedthat silver ions, menthol and TWEEN 20, all act in synergy, thus furtherallowing to beneficially utilize low concentrations of silver ions inthe antiseptic composition while exhibiting the desired activity.

The antiseptic composition described herein can further compriseadditional ingredients, which are aimed at improving or facilitating itspreparation, application and/or performance. Such additional ingredientsinclude, for example, anti-irritants, anti-foaming agents, humectants,deodorants, antiperspirants, pH adjusting agents, preservatives,emulsifiers, occlusive agents, emollients, thickeners, penetrationenhancers, colorants, propellants (depending on the final form of thecomposition) and surfactants.

Representative examples of humectants that are usable in this context ofthe present invention include, without limitation, guanidine, glycolicacid and glycolate salts (e.g. ammonium slat and quaternary alkylammonium salt), aloe vera in any of its variety of forms (e.g., aloevera gel), allantoin, urazole, polyhydroxy alcohols such as sorbitol,glycerol, hexanetriol, propylene glycol, butylene glycol, hexyleneglycol and the like, polyethylene glycols, sugars and starches, sugarand starch derivatives (e.g., alkoxylated glucose), hyaluronic acid,lactamide monoethanolamine, acetamide monoethanolamine and anycombination thereof.

Suitable pH adjusting agents include, for example, tris-imidazoletrimethylamine buffers, and any other buffer solutions compatible withthe other components without causing precipitation of any of thecomponents.

Representative examples of deodorant agents that are usable in thecontext of the present embodiments include, without limitation,2,4,4′-trichloro-2′-hydroxy diphenyl ether, and diaminoalkyl amides suchas L-lysine hexadecyl amide.

Suitable preservatives that can be used in the context of the presentembodiments include, without limitation, one or more alkanols, parabenssuch as methylparaben and propylparaben, propylene glycols, sorbates,urea derivatives such as diazolindinyl urea, or any combinationsthereof.

Suitable emulsifiers that can be used in the context of the presentembodiments include, for example, one or more sorbitans, alkoxylatedfatty alcohols, alkylpolyglycosides, soaps, alkyl sulfates, or anycombinations thereof.

Suitable occlusive agents that can be used in the context of the presentembodiments include, for example, petrolatum, mineral oil, beeswax,silicone oil, lanolin and oil-soluble lanolin derivatives, saturated andunsaturated fatty alcohols such as behenyl alcohol, hydrocarbons such assqualane, and various animal and vegetable oils such as almond oil,peanut oil, wheat germ oil, linseed oil, jojoba oil, oil of apricotpits, walnuts, palm nuts, pistachio nuts, sesame seeds, rapeseed, cadeoil, corn oil, peach pit oil, poppyseed oil, pine oil, castor oil,soybean oil, avocado oil, safflower oil, coconut oil, hazelnut oil,olive oil, grape seed oil and sunflower seed oil.

Suitable emollients, that can be used in the context of the presentembodiments include, for example, dodecane, squalane, cholesterol,isohexadecane, isononyl isononanoate, PPG Ethers, petrolatum, lanolin,safflower oil, castor oil, coconut oil, cottonseed oil, palm kernel oil,palm oil, peanut oil, soybean oil, polyol carboxylic acid esters,derivatives thereof and mixtures thereof.

Suitable thickeners that can be used in the context of the presentembodiments include, for example, non-ionic water-soluble polymers suchas hydroxyethylcellulose (commercially available under the TrademarkNatrosol® 250 or 350), cationic water-soluble polymers such as Polyquat37 (commercially available under the Trademark Synthalen® CN), fattyalcohols, and mixtures thereof.

Suitable penetration enhancers usable in context of the presentembodiments include, but are not limited to, polyethylene glycolmonolaurate (PEGML), propylene glycol (PG), propylene glycol monolaurate(PGML), glycerol monolaurate (GML), lecithin, the 1-substitutedazacycloheptan-2-ones, particularly 1-n-dodecylcyclazacycloheptan-2-one(available under the trademark Azone® from Whitby Research Incorporated,Richmond, Va.), alcohols, menthol, TWEENS such as TWEEN 20, and thelike. The permeation enhancer may also be a vegetable oil. Such oilsinclude, for example, safflower oil, cottonseed oil and corn oil.

Suitable anti-irritants that can be used in the context of the presentembodiments include, for example, steroidal and non steroidalanti-inflammatory agents or other materials such as menthol, aloe vera,chamomile, alpha-bisabolol, cola nitida extract, green tea extract, teatree oil, licoric extract, allantoin, caffeine or other xanthines,glycyrrhizic acid and its derivatives.

Any of the additional ingredients or agents described herein ispreferably selected as being compatible with the silver ions, at leastwithin the concentration range it is used within the composition, suchthat no precipitation occurs and there is no interference with theavailability of the silver ions in the composition.

Any of the additional ingredients described herein is further preferablyselected as being biocompatible.

It is noted that some agents or ingredients included within thecomposition may provide a dual effect. For example, menthol is used toact in synergy with silver ions for enhancing an antiseptic activity,but is also useful as a penetration enhancer and as an anti-irritant.TWEEN 20 can be used as a solubilizing agent but is also known to act asa penetration enhancer.

In some embodiments, the antiseptic composition further comprises anadditional therapeutically active agent, for example, an agent capableof treating the indicated condition, as detailed herein, or an agentcapable of disinfecting a surface, as is further detailed hereinbelow(e.g., a bodily surface). In some embodiments, the antisepticcomposition further comprises an agent capable of preventing, reducingor inhibiting a growth of a disinfecting microorganism e.g. bacteria orfungi, or an agent capable of reducing a load of a disinfectingmicroorganism.

In some embodiments, the antiseptic composition further comprises anadditional agent that is capable of treating a wound, as describedherein. Exemplary additional therapeutically active agents include, butare not limited to, betaine and polyhexadine.

The antiseptic composition described herein is formulated together witha pharmaceutically acceptable and suitable carrier.

As used herein, the term “pharmaceutically acceptable carrier” describesa carrier or a diluent that is used to facilitate the administration ofthe composition and which does not cause significant irritation to anorganism and does not abrogate the biological activity and properties ofthe administered active compounds. Examples, without limitations, ofcarriers include water, buffered aqueous solutions, propylene glycol,emulsions and mixtures of organic solvents with water, as well as solide.g. powdered) and gaseous carriers.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Antiseptic compositions for use in accordance with the presentembodiments thus may be formulated in conventional manner using one ormore pharmaceutically acceptable carriers, excipients and/orauxiliaries, which facilitate processing of the compounds intopreparations which can be used pharmaceutically. The dosage may varydepending upon the dosage form employed and the route of administrationutilized.

The exact formulation, route of administration and dosage can be chosenby the individual physician in view of the patient's condition (seee.g., Fingl et al., 1975, in “The Pharmacological Basis ofTherapeutics”, Ch. 1 p. 1).

The pharmaceutically acceptable carrier can be either an organic carrieror an aqueous carrier. In some embodiments, the carrier is an aqueouscarrier, in which the source of silver ions is soluble. In someembodiments, the aqueous carrier is a buffer solution.

An aqueous carrier preferably comprises injectable-grade water, i.e.,USP grade “water for injection”. However, other forms of purified watermay be suitable, such as, for example, distilled and deionized water.

Aqueous formulations are preferred since these formulations are gentleto both skin and mucosal tissue and are suitable for use on open wounds.However, non-aqueous formulations are also contemplated. For example, incases where the antiseptic composition is in a form of a paste or anemulsion, non-aqueous carriers or mixed carriers of aqueous and organiccarriers can be used, as long as silver ions are generated in thecarrier.

The antiseptic composition may be formulated for administration ineither one or more of routes, depending on the area to be treated.

According to some embodiments, the antiseptic composition is formulatedfor topical application, as a topical dosage form.

As used herein, the phrase “topical dosage form” describes a dosage formsuitable for topical administration to the treated area.

By selecting the appropriate carrier and optionally other ingredientsthat can be included in the composition, the antiseptic compositionsdescribed herein may be formulated into any form normally employed fortopical application. Hence, the compositions described herein can be,for example, in a form of a cream, an ointment, a paste, a gel, alotion, a milk, a solution, an aerosol, a spray, a foam, a gauze, awipe, a sponge, a non-woven fabric, a cotton fabric, a pledget, a patchand a pad.

Exemplary topical dosage forms include, but are not limited to, a cream,a spray, a gauze, a wipe, a sponge, non-woven fabrics, a cotton fabrics,a foam, a solution, a lotion, an ointment, a paste and a gel.

Such topical dosage forms may optionally further comprise an adhesive,for facilitating the topical application of the composition onto thetreated area for a prolonged time period.

In some embodiments, the antiseptic composition is formulated as aliquid reservoir, to be applied as drops, spray, aerosol, liquid, foamand the like. Suitable carriers and other ingredients are used in thesecases. For example, for application as an aerosol or foam, a propellantis used.

In some embodiments, the antiseptic composition is formulated as acream. An exemplary cream formulation can be obtained by mixing theantiseptic composition described herein with a carrier comprisingcellulose derivatives such as cellulose acetate, hydroxyethyl celluloseand/or a polyethylene glycol.

The amount of a composition to be administered will be, of course,dependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA (the U.S. Food and DrugAdministration) approved kit, which may contain one or more unit dosageforms containing the active ingredient. The pack may comprise, forexample, glass or plastic foil. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied by a notice associated with the container in aform prescribed by a governmental agency regulating the manufacture, useor sale of pharmaceuticals, which notice is reflective of approval bythe agency of the form of the compositions for human or veterinaryadministration. Such notice, for example, may be of labeling approved bythe U.S. Food and Drug Administration for prescription drugs or of anapproved product insert. Compositions of the invention formulated in acompatible pharmaceutical carrier may also be prepared, placed in anappropriate container, and labeled for disinfection purposes or fortreatment of wounds, as detailed herein.

The compositions described herein may be packed or presented in anyconvenient way. For example, they may be packed in a tube, a bottle, adispenser, a squeezable container, or a pressurized container, usingtechniques well known to those skilled in the art and as set forth inreference works such as Remington's Pharmaceutical Science 15^(th) Ed.It is preferred that the packaging is done in such a way so as tominimize contact of the unused compositions with the environment, inorder to minimize contamination of the compositions before and after thecontainer is opened.

The compositions described herein are preferably supplied in theconcentration intended for use but may also be prepared as concentratesthat are diluted prior to use. For example, concentrates requiringdilution ratios of 2:1 to 10:1 parts carrier (e.g., water) toconcentrate are contemplated. The higher limit of the concentrate islimited by the solubility and compatibility of the various components athigher concentrations.

In some embodiments, the antiseptic composition described herein ispackaged in a packaging material and identified in print, in or on thepackaging material, for use in disinfecting a surface, as describedherein.

In some embodiments, the antiseptic composition described herein ispackaged in a packaging material and identified in print, in or on thepackaging material, for use in disinfecting a bodily surface, as isfurther detailed hereinbelow.

In some embodiments, the antiseptic composition is packaged in apackaging material and identified in print, in or on the packagingmaterial, for use in the treatment of wounds.

In some embodiments, the antiseptic composition is packaged in apackaging material and identified in print, in or on the packagingmaterial, for use in the treatment of an infection, as is furtherdetailed hereinbelow.

The efficacy of the compositions described herein as disinfectants andin treating wounds is well demonstrated in the Examples section thatfollows.

Accordingly, according to an additional aspect of embodiments of theinvention there is provided a kit, which comprises the antisepticcomposition described herein, being packaged in a packaging material.

The kit can be labeled, for example, by being identified in print, in oron the packaging material, for use for disinfection purposes and/or fortreating wounds, as detailed herein.

The components of the antiseptic composition can be packaged within thekit either together, as a single composition, or at least one of thecomponent can be packaged individually. When one or more components arepackaged individually, the kit may further be supplied with instructionsindicating the route of preparing an antiseptic composition ready foruse. Such instructions can be, for example, in accordance with theprocess of preparing the antiseptic composition, as is described indetail hereinafter.

According to a further aspect of some embodiments of the presentinvention, there is provided a method of disinfecting a substrate, whichis effected by applying an effective amount of the antisepticcomposition described herein onto the substrate, thereby disinfectingthe substrate.

As used herein throughout, the term “disinfecting” refers to reducingthe load of microorganism(s) in or on the substrate, to decreasing therate of microorganism(s) growth in or on the substrate, to inhibiting orpreventing the growth of microorganism(s) in or on the substrate, and/orto eradicating microorganism(s) in or on the substrate.

The term “substrate” as used herein, refers to any structure, product ormaterial which can undesirably support, harbor or promote the growth ofa microorganism on a surface thereof. Non-limiting examples includemedical devices, medical equipments, containers of medical andpharmaceutical products, packaging materials, industrial equipments andmachines used in the pharmaceutical, medical, agricultural,nutraceutical and food industries, walls, buildings, warehouse,compartment, container or transport vehicle, a dye or a paint and anyother materials and industrial compounds which require protection oftheir surfaces against microbial attacks, such as, for example,construction materials.

According to some embodiments, applying the antiseptic composition ontothe substrate is effected by, for example, washing the substrate withthe composition, spreading the composition onto the substrate's surface,spraying the composition onto the substrate, or dipping the substrate inthe composition. The route of application depends, at least in part, onthe composition's form.

According to some embodiments, the substrate is a bodily surface of asubject in need thereof.

As used herein, the term “subject” refers to any animal e.g., a mammal,including, but not limited to, humans, non-human primates, mammals,rodents, and any other animal, which is to be the recipient of aparticular treatment. Typically, the terms “subject” and “patient” areused interchangeably herein in reference to a human subject.

According to some embodiments, the method is effected by topicalapplication of the antiseptic composition onto the bodily surface.According to some embodiments, the bodily surface is skin, a skintissue, a mucosal tissue or any other infected or potentially infectedbody area that can benefit from topical application.

Accordingly, an antiseptic composition as described herein can beutilized for disinfecting an infected area (e.g., an acute or chronicwound, as detailed hereinafter) or for preventing infections in areasthat are at risk for being infected. The latter include, for example,surgical wounds, acute wounds, ulcers and the like, which are highlysusceptible to infections by various microorganisms.

As discussed hereinabove, the antiseptic compositions described hereinhave demonstrated efficient bactericidal activity against variousbacterial strains as well as efficient fungicidal activity againstvarious fungal strains. Thus, the antiseptic compositions describedherein can be utilized in the treatment of wounds where the reduction ofthe microbial load in the wound is therapeutically beneficial.

Thus, according to an aspect of some embodiments of the presentinvention, there is provided a method of treating a wound in a subjectin need thereof, the method comprising applying an effective amount ofthe antiseptic composition described herein to the wound area, therebytreating the wound.

According to some embodiments, the method is effected by topicallyapplying the antiseptic composition onto the wound area.

The term “wound” is used herein to be construed according to itsbroadest meaning so as to describe damaged or disturbed skin or mucosalarea, whether or not containing devitalized or eschar tissue, andencompasses any type of wound, including, but not limited to, acutewounds, chronic wounds, surgery wounds, burns and the like. The term“skin lesion” as used herein describes damaged skin and is usedinterchangeably with the term “wound” throughout the application.

The term “wound area” describes the area adjacent to the wound. Thisarea typically extends from immediately adjacent the wound up to about30 cm. It is being understood that the inner boundary of the areaperipheral to the wound may conform to or parallel the shape of thewound.

The term “acute wound” describes a wound caused by a traumatic abrasion,laceration or through superficial damage, and which eventually healsspontaneously without complications through normal phases of woundhealing (such as hemostasis, inflammation, proliferation andremodeling). Acute wounds, however, can often be complicated if becomein contact with pathogenic microorganisms that may lead to localinfection.

The phrases “surgery wound” and “surgical wound”, which are used hereininterchangeably, describe a wound that is formed as a result of asurgical procedure. Surgical wound infections are common, being 12% ofall hospital-acquired infections. The rate of infection varies dependingon the type of surgery undertaken. Especially high rates are associatedwith contaminated surgery, such as colorectal surgery or delayed surgeryto traumatic wounds. Surgical wound infections are usually caused by thepatient's normal flora or by bacteria from the environment or the skinof hospital staff. The most common microorganism that leads to surgicalwould infection is Staphylococcus aureus. Other common causativemicroorganisms include other Gram-negative aerobes, Streptococcus spp.and anaerobes.

The term “burns” describes wounds caused by heat, cold, electricity,chemicals, light, radiation, or friction. Burns can be highly variablein terms of the tissue affected, the severity, and resultantcomplications. Muscle, bone, blood vessel, and epidermal tissue can allbe damaged with subsequent pain due to profound injury to nerve endings.Depending on the location affected and the degree of severity, a burnvictim may experience a wide number of potentially fatal complicationsincluding shock, infection, electrolyte imbalance and respiratorydistress. Infection is the most common complication of burns and is themajor cause of death in burn victims. More than 10,000 Americans dieevery year from infectious complications of burns. Pathogenicmicroorganisms that commonly infect burn wounds include, for example,gram-positive bacteria such as methicillin-resistant Staphylococcusaureus (MRSA) and gram-negative bacteria such as Acinetobacterbaumannii-calcoaceticus complex, Pseudomonas aeruginosa, and Klebsiellaspecies.

The phrase “chronic wound” describes a wound in which there is no clotformation, normally occurring in patients who are compromised in somefashion and are less likely to heal. Examples of chronic wounds arechronic cutaneous ulcers such as diabetic ulcers, decubitus ulcers(pressure ulcers), and venous ulcers.

The phrase “diabetic ulcers” describes a wound caused by combination offactors associated with diabetes, such as decreased circulation, loss ofsensation, structural foot deformities and loss of skin integrity. Adiabetic ulcer can be a simple break in the skin, which does not heal ina timely and orderly fashion, or a wound that extends to deep structuresand bone. Diabetic ulcers are often the entry points for bacteria andfungal organisms to invade the body, and the cause of limb and lifethreatening infection, often referred to in the art as diabeticinfection. Diabetic infections are usually polymicrobial involvinginfections caused by multiple aerobic and anaerobic microorganisms.Staphylococcus aureus, beta-hemolytic streptococcus, Enterobacteriaceae,Bacteroides fragilis, Peptococcus, and Peptostreptococcus are exemplarystrains that were cultured from diabetic ulcers.

The phrase “decubitus ulcers”, also known as “pressure ulcers”, describeskin lesions caused by variable factors such as: unrelieved pressure;friction; humidity;

shearing forces; temperature; age; continence and medication; to anypart of the body, especially portions over bony or cartilaginous areassuch as sacrum, elbows, knees, ankles etc. Although easily prevented andcompletely treatable if found early, decubitus ulcers are often fataland are one of the leading iatrogenic causes of death reported indeveloped countries. Decubitus ulcers may be caused by inadequate bloodsupply and resulting reperfusion injury when blood re-enters tissue. Themost common organisms isolated from pressure ulcers are Proteusmirabilis, group D streptococci, Escherichia coli, Staphylococcusspecies, Pseudomonas species, and Corynebacterium organisms.

The phrase “venous ulcer” describes wounds that are thought to occur dueto improper functioning of valves in the veins, usually of the legs,causing the pressure in the veins to increase. They are a major cause ofchronic wounds, occurring in approximately 30-40% of chronic woundcases. Most venous ulcers are heavily contaminated with bacteria such asStaphylococcus, Eschrichia coli, Proteus and Pseudomonas.

As discussed hereinabove, reduction of the bacteria cell load in acutewounds and chronic wounds such as diabetic ulcers, decubitus ulcers(pressure ulcers), venous ulcer as well as burns by the antisepticcompositions described herein is therapeutically beneficial.

As further discussed hereinabove, the antiseptic compositions describedherein have been found to effectively inhibit the growth of variousbacterial strains. For example, a solution of 0.01% silver ions and0.05% menthol, a composition according to some embodiments of thepresent invention, has been shown to efficiently kill upon contact andprevent growth of the bacterial strains Escherichia coli ATCC 47076,Klebsiella pneumoniae, a Methicillin Resistant clinical strain ofStaphylococcus aureus (MRSA), a clinical strain of Staphylococcusepidermis, a clinical strain of Extended Spectrum β-lactamase producingEscherichia coli, a clinical strain of Multi Drug ResistantAcinetobacter baumannii, and a clinical strain of Multi Drug ResistantPseudomonas aeruginosa (see, Tables 3 and 4). A solution of 0.01% silverions and 0.05% menthol, a composition according to some embodiments ofthe present invention, has further been shown to exhibit fungicidalactivity against Trichophyton rubrum clinical strain and Microsporumcanis clinical strain (see, Example 6 hereinafter and FIGS. 2-4).Therefore, due to their broad antiseptic activity against variousbacterial and fungal strains, including strains which are known to beisolated from wound areas, the antiseptic compositions described hereinmay be efficiently utilized in the treatment of wounds.

As further discussed herein and is demonstrated in the Examples sectionthat follows, the antiseptic compositions described herein were shown toeffectively inhibit microorganism's growth and to effectively preventgrowth of various microorganisms in a non-sterile sample.

Accordingly, the antiseptic compositions described herein can be used ina method of treating or preventing an infection. The infection can be abacterial infection, a fungal infection, an infection caused by a yeast,or any combination thereof.

The antifungal activity of the composition containing silver ions andmenthol as described herein carries a vast therapeutic potential withinthe context of chronic diabetic foot ulcers as well as within thecontext of antifungal treatments of onychomycosis (fungal nailinfections, mainly caused by the dermatophyte Trichophyton rubrum),dermatophytosis such as Tinea pedis (athlete's foot, also affected byTrichophyton rubrum) and Tinea corporis (dermatophyte skin invasion) andTinea capitis (ringworm of the scalp and dermatophyte hair invasion)mainly affected by Microsporum canis.

There is a significant association between diabetes and occurrence offungal foot infections: while in non-diabetic elderly population theincidence is 60%, in diabetic subjects the incidence rises to over 80%.Diabetes mellitus has a significant adverse effect on the occurrence ofboth Tinea pedis and onychomycosis, affecting x 1.48 increase ratio.Onychomycosis, often associated with Tinea pedis, substantially increasethe risk of other infections, lesions formation eventually leading tochronic ulcers and amputations. The incidence of Tinea pedis in diabeticpatients is 32% vs 7% in control population [Gupta A K, Humke S (2000)Eur J Dermatol 10: 379-384; Saunte et al. (2006) Acta Derm Venereol86:425-428].

The term “disinfecting”, as used herein throughout, thus encompassestreating an infection, as described herein.

In addition, the antiseptic compositions described herein can also beused as preservatives, for preventing or reducing formation of microbialload under non-sterile conditions and/or under conditions suspected asbeing adversely non-sterile.

The antiseptic compositions described herein can therefore be added, forexample, to storage containers, particularly storage containers forstoring and/or transporting medical products, such as medical devices,pharmaceutically active agents and drugs.

According to further aspects of embodiments of the invention, there isprovided a use of the antiseptic composition described herein in themanufacture of a product for disinfecting a substrate, as describedherein.

According to further aspects of embodiments of the invention, there isprovided a use of the antiseptic composition described herein in themanufacture of a medicament for disinfecting a surface of a bodily areaand/or for treating wounds, as described herein.

In any of the methods and uses described herein, the antisepticcompositions can be applied to the treated area using any suitablemeans.

Ordinarily, an absorbent of some type such as gauze, foam sponges,non-woven fabrics, cotton fabrics, cotton swabs or balls, and the like,are soaked with the composition and applied over the wound.

Alternatively, the composition can be in a form of a spray or aerosol,which can further comprise, for example, a film-forming agent. Thecomposition is thus applied on the treated area by spraying.

Further alternatively, the composition can be in a form of a cream, apaste or a gel, and is applied onto the wound by spreading, or byapplying an adhesive patch that comprises the composition.

Further alternatively and preferably, higher rates of wound healing canbe achieved by applying the antiseptic composition over and through thewound in continuous flow so that the concentrations of the antisepticagents and the hyperosmotic agent in contact with the wound are keptconstant.

Accordingly, in some embodiments of the invention, the topicalapplication of the antiseptic composition described herein is performedby streaming a flow of the antiseptic composition over and through thewound area.

The flow of the compositions described herein is conveyed over andthrough the wound for a predetermined period of time. According to someembodiments, conveying the flow continuously is performed for at least 5minutes, in some embodiments for at least 15 minutes, in someembodiments for at least 30 minutes and in some embodiments for at least1 hour. The wound is monitored for assessing the progress of reducingthe microbial load and wound healing. A pause in the treatment for shortperiods of time can be carried out by arrest of the flow and removal ofany conduits that prevent a free movement of the patient. An occlusivedressing to which the conduits are reattached may be left over thewound.

Treatment can be applied using an apparatus as described in U.S. patentapplication having Publication No. 2004/0186421 or WO2005/070480, whichare incorporated by reference as if fully set forth herein.

According to an exemplary embodiment, such an apparatus comprises ahousing having at least one aperture formed therein and means foraffixing the apparatus to the skin around the circumference of the skinlesion, wherein the housing comprises (i) at least one inlet tube havinga first longitudinal axis and configured to be adjustable along itslongitudinal axis through the aperture; and (ii) at least one outlettube having a second longitudinal axis.

The apparatus further comprises a reservoir adapted for holding theantiseptic composition, the reservoir being in fluid communication withthe one or more inlets. An outlet may comprise valves for controllingthe flow between the reservoir and the treatment zone. Preferably, anoutlet may further comprise means enabling disconnecting andreconnecting the housing from the reservoir, thereby enabling to pausethe treatment.

The simplicity of disconnecting and reconnecting the patient from theflow means enables applying the antiseptic compositions for prolongedperiods of time. A flow of the antiseptic composition may last for atleast one hour or may last for several hours, depending on individualneeds.

According to some embodiments, the flow is induced by gravity from atleast one reservoir that comprises the antiseptic composition.

According to some embodiments, the flow is induced by a pump being influid communication with at least one reservoir that comprises theantiseptic composition.

In some embodiments, the flow is induced by a peristaltic pump, which isnot in direct contact with the streamed solution.

According to a further aspect of embodiments of the invention there isprovided a process for preparing the antiseptic composition describedherein, the process comprising mixing the source of silver ions, thementhol and the pharmaceutically acceptable carrier, so as to obtain theantiseptic composition.

According to some embodiments, the process further comprises admixing ahyperosmotic agent with the composition.

The order of addition of the composition components (i.e. source ofsilver ions, menthol, a pharmaceutically acceptable carrier andoptionally a hyperosmotic agent) may vary, depending, for example, onsolubilization considerations.

Thus, for example, the process can be effected by preparing a solutionof a source of silver ions and carrier, and adding thereto menthol.Alternatively, the process can be effected by preparing a solution ofmenthol and a carrier and adding thereto the source of silver ions.

The hyperosmotic agent, if present, can be added before or after theaddition of any of the silver ions or the menthol. Similarly, any otheringredients that are added to the composition, as described hereinabove,can be added at any stage.

According to some embodiments the process further comprises admixingsaid menthol with a solubilizing agent such as TWEEN 20, as describedherein, to thereby obtain a solution of menthol and the solubilizingagent; and admixing said solution of menthol with the composition.

In some embodiments, the solubilizing agent is TWEEN 20. In someembodiments, the weight ratio between the menthol and the TWEEN 20 is1:10. In some embodiments, the TWEEN 20 is utilized at a concentrationof 0.5% w/v based on the total volume of the menthol and TWEEN 20solution.

In some embodiments, the amounts of the source of silver ions and thementhol are such that upon mixing, the final concentration of the silverions is from 0.05 mM to 30 mM, and the menthol final concentration isfrom 0.3 mM to 32 mM w. In some embodiments, the final concentration ofthe source of silver ions is from 0.3 mM to 0.6 mM, and the mentholfinal concentration is from 0.6 mM to 6.4 mM.

Considering the synergistic effect exhibited by combining menthol and asource of silver ions, which allows using a substantially reducedconcentration of silver ions, as compared to currently availableantiseptic products, according to a further aspect of embodiments of theinvention there is provided a method of reducing a concentration ofsilver ions in an antiseptic composition which comprises silver ions (asan active ingredient). The method, according to these embodiments, iseffected by admixing with a source of silver ions, a synergisticallyeffective amount of menthol.

In some embodiments, the concentration of the silver ions is reduced byat least 2-folds, at least 3-folds, at least 5 folds, at least 6 folds,at least 7-folds, at least 8-folds, at least 9-folds, and even by anorder of magnitude, and even twice thereof, or by 30-folds, 40 folds,50-folds, 60-folds, 70-folds, 80-folds, 90-folds and 100-folds, ascompared to an antiseptic composition that is devoid of asynergistically effective amount of menthol.

In some embodiments, the concentration of the silver ions is reduced bya 50-folds, as compared to an antiseptic composition that is devoid of asynergistically effective amount of menthol.

The phrase “antiseptic effective amount” describes the amount of anagent, herein, a source of silver ions, which exhibits an antisepticactivity, as defined herein.

The phrase “synergistically effective amount” describes a concentrationof menthol upon being admixed with the source of silver ions in theantiseptic composition, which results in a synergistic effect of thecombination of silver ions and menthol, as defined herein.

Further according to embodiments of the invention, there is provided amethod of increasing an antiseptic activity of an antiseptic compositionwhich comprises a source of silver ions at a concentration that is lowerthan 6 mM, the method being effected by admixing with the antisepticcomposition a synergistically effective amount of menthol.

Admixing menthol with the antiseptic composition can be effected inaccordance with the process of preparing an antiseptic composition, asdescribed herein. Accordingly, additional components, such as ahyperosmotic agent, and a solubilizing agent, can also be admixed withthe silver ions.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimental[support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Materials and Experimental Methods

Materials:

LB medium was prepared by dissolving 10 grams Tryptone (Cat. No. 161200,Pronadisa, Conda), 5 grams yeast extract (Cat. No. 212750, DIFCO), 10grams NaCl (Cat. No. 1.06404.1000, Merck) and 2 grams glucose (Cat. No.1.08337.1000, Merck) in a final volume of 1 liter of highly purifiedwater with magnetic stirring and mild heating. LB-agar was prepared bythe addition of 15 grams of Bacto agar (Cat. No. 214010, BD) to LBmedium.

BHI growth medium was obtained from Laboratory of Molecular Epidemiologyand Antimicrobial Resistance, Tel Aviv Sourasky Medical Center.

NaNO₃ was obtained from Riedel-deHaen (Cat. No. 31440).

TWEEN 20 (Cat. No. 8.17072.1000), AgNO₃ (Cat. No. 1.01510.0050), Menthol(Cat. No. 1.05995.1000) Glycerol (Cat. No. 1.04093.1000), Tris (Cat. No.1.08386.1000) and acetic acid glacial (Cat. No. 1.00056.2500) wereobtained from Merck.

Compositions:

Silver nitrate and menthol solutions were prepared by adding finalconcentrations of 10% glycerol (hyperosmotic agent), menthol (0.5%TWEEN-20) and silver nitrate to a 50 mM Tris-buffer solution. The finalcomposition was tittered to pH 7.45 using acetic acid glacial and thenfiltered through a 0.22 μm PES membrane filter. The sterile solutionsthus obtained were clear and devoid of participates.

General Protocols:

E. Coli Growth Protocol:

A frozen stock of E. coli ATCC 47076 was grown overnight (1 ml in 9 mlLB medium) in a shaker incubator (200 rpm at 37° C.) until theOD_(600nm) of the medium reached 1.6-1.8. One-ml aliquots of the culturemedium were then transferred into 50 ml of fresh sterile LB medium andplaced in a shaker incubator (200 rpm at 37° C.) for two hours until theOD_(600nm) of the medium reached 0.6-0.8 (2×10⁸ colony forming unitesper ml (CFU/ml)). The Bacterial suspension was adjusted to 10⁸ CFU/ml bydilution with sterile medium. The Bacterial cells were then washed threetimes with 0.9% NaNO₃. The NaNo₃ washing cycles was performed bytransferring 5 ml of the bacterial suspensions into 15 ml test tubes andsubjecting the suspension to centrifugation at 4000 rpm at 4° C. for 5minutes, followed by removal of the supernatant and re-suspension of thebacterial cell pellet in 5 ml of 0.9% NaNO₃.

The bactericidal activity of various concentration combinations ofsilver ions (from AgNO₃) and menthol (solubilized by the addition of0.5% TWEEN 20) was then assessed by adding 5 ml of the testedcombinations to the bacterial cell pellets prepared as describedhereinbelow and re-suspending the cell pellets by vortex. A test tubecontaining only 0.9% NaNO₃ served as control.

The bacterial suspensions thus obtained were transferred to sterileglass tubes to reduce potential cell adsorption to the walls, and keptcovered at 37° C. throughout the testing period. Following 5 minutes ofstatic incubation, 100 μl samples of the treated bacterial suspensionwere removed, and 100 of decimal dilutions in NaNO₃ 0.9% of thesuspension were dropped onto LB agar plates. The level of bacterialproliferation was assessed by counting the number of colonies that grewon the plates and calculating the CFU/ml and log₁₀ CFU/ml following 18hours of incubation at 37° C. Higher log₁₀ CFU/ml calculated for abacterial suspension, incubated with a specific combination, isindicative of a lower level of bactericidal activity of thatcombination.

Growth Protocol of Other Bacteria Cell Lines:

Various bacterial cells lines were obtained from the clinical strainscollection of the Laboratory of Molecular Epidemiology and AntimicrobialResistance, Tel Aviv Sourasky Medical Center. The bactericidal activityof the tested antiseptic combinations was thus tested against Klebsiellapneumoniae, a Methicillin Resistant clinical strain of Staphylococcusaureus (MRSA), a clinical strain of Staphylococcus epidermis, a clinicalstrain of Extended Spectrum β-lactamase producing Escherichia coli, aclinical strain of Multi Drug Resistant Acinetobacter baumannii, and aclinical strain of Multi Drug Resistant Pseudomonas aeruginosa.

The bacterial growth protocol was essentially as described above onlythat bacteria cell lines were grown in BHI growth medium (and followingthe growth of the cells in the medium, the bacterial suspension wasadjusted to 10⁷ CFU/ml by dilution with sterile medium). In addition, inthese experiments, the static incubation of each bacterial suspensionwith the silver ion+menthol combination was for 0, 30 and 60 minutes.

Synergy:

To assess whether menthol and silver ions act synergistically, thebactericidal activity observed when menthol and silver ions wereadministered together was compared to the bactericidal activity observedwhen each of these compounds was administered alone (determined afterLehmann, 2000. Synergism in Disinfectant Formulation, in Disinfection,Sterilization, and Preservation 5th ed., S. S. Block, Editor. LippincottWilliams and Wilkins. pp. 459-472).

A combination was considered synergistic when:

The bactericidal activity of menthol and silver ions was larger than theadditive value of the bactericidal activity observed when each compoundwas tested, at the specific concentration, alone; or

The time, until a certain level of bactericidal activity of menthol andsilver ions was detected, was shorter than the time measured when eachagent was tested, at the specific concentration, alone.

Example 1 Synergism of Bactericidal Activity of Menthol and Silver Ionsin a Hyperosmotic Solution of 10% (v/v) Glycerol on E. coli ATCC 47076

For evaluation of synergy in bactericidal activity between menthol andsilver ions, various concentration combinations of silver ions andmenthol were used, as detailed in Table 1.

The results are summarized in Table 1 and show that synergy was observedfor silver ions (derived from AgNO₃) and menthol combinations of 0.005%(w/v)+0.1% (w/v), 0.005%+0.05%, 0.0075%+0.05%, 0.01%+0.05% and0.01%+0.01% w/v concentrations, respectively (corresponding to theshadowed areas in Table 1). These concentrations correspond to silverions and menthol combinations at the following combinations: 0.29 mM+6.4mM, 0.29 mM+3.2 mM, 0.44 mM+3.2 mM, 0.6 mM+3.2 mM and 0.6 mM+0.64 mM.

The left part of Table 1 presents the observed percentage of bacterialcells killed by each combination whereas the right part of Table 1presents the expected percentage of bacterial cells killed if thebactericidal activity of menthol and silver ions were additive in naturerather than synergistic.

TABLE 1

^(a)percentage of E.Coli cells killed by exposure to the indicatedcombination of menthol and AgNO₃. ^(b)expected percentage of E. Colicells killed, by the indicated combination when assuming an additiverather than synergistic activity between menthol and silver ions.

The synergistic combination of 0.01% silver ions and 0.05% menthol wasselected for further studies.

Example 2 Bactericidal Activity of a Combination of 0.01% (w/v) SilverIons and 0.05% (w/v) Menthol in a Hyperosmotic Solution Containing 10%(v/v) Glycerol on Various Types of Bacteria

The effect of a combination of 0.01% silver ions and 0.05% menthol(solubilized by 0.5% (v/v) TWEEN 20) on the growth and viability ofvarious types of bacteria was evaluated.

Cells incubated with NaNO₃ 0.9% (w/v) solution alone, without theaddition of any antiseptic agent, served as control.

Bactericidal activity was defined as a 5-log₁₀ or lower reduction incolony count as compared to control (after Cremieux, Methods of testingDisinfectants, in Disinfection, Sterilization, and Preservation 5th ed.,S. S. Block, Editor. 2000, Lippincott Williams and Wilkins, pp1305-1328.

Table 2 presents the log₁₀ CFU/ml calculated for different types ofbacterial cell suspensions incubated for 0, 30 and 60 minutes with noantiseptic agent (control) or either 0.01% silver ions (0.01% AgNO₃)alone, 0.05% menthol alone or a combination of 0.01% silver ions and0.05% menthol. A baseline level was defined as the observed bacteriagrowth at time 0 when no antiseptic agent was added to the bacterialsuspension.

The results clearly demonstrate the complete eradication of bacteriaviability upon incubation of the bacterial suspension with thecombination of 0.01% silver ions and 0.05% menthol. The effect wasevident immediately (at time point 0) with the only exception being inthe case of a Methicillin Resistant clinical strain of Staphylococcusaureus in which case an immediate 50% reduction in viability wasobserved followed by the killing of all bacteria after 30 minutes.

TABLE 2 0.01% AgNO₃ 0.01% 0.05% and 0.05% Control^(a) AgNO₃ ^(a)Menthol^(a) Menthol^(a) Bacteria Baseline 0 30 60 0 30 60 0 30 60 0 3060 Escherichia coli 7.3 7.3 7.3 7.3 6.4 0 0 5.3 0 0 0 0 0 ATCC 47076Klebsiella 6.8 6.8 6.7 6.5 0 0 0 6.2 4.9 3.8 0 0 0 pneumoniaeStaphylococcus 6.1 6.1 6.1 6.1 5.4 0 0 6.3 6.3 6.2 3.4 0 0 aureusStaphylococcus 5.4 5.4 5.3 5.2 0 0 0 5.4 5.4 5.3 0 0 0 epidermisbeta-lactamase 5.9 5.9 5.8 5.6 4.8 0 0 4.9 4.9 4.9 0 0 0 producingEscherichia coli Acinetobacter 6.4 6.4 6.4 6.4 6.3 0 0 6.4 6.3 6.2 0 0 0baumannii Pseudomonas 6.5 6.5 6.5 7.1 5.1 0 0 5.8 3.5 2.5 0 0 0aeruginosa ^(a)log₁₀ CFU/ml of bacteria when the bacterial suspensionswere treated with the indicated treatment for 0, 30 and 60 minutes.

These results point to a synergistic bactericidal activity exhibited bycombining silver ions and menthol.

Example 3 Determination of the Minimum Inhibitory Concentration (MIC)and the Minimum Bactericidal Concentration (MBC) of a Combination of0.01% (w/v) Silver Ions and 0.05% (w/v) Menthol

General:

Studies were conducted for determining the effective use dilution of thetested silver ions-menthol combination against the test organism(s)using a tube dilution method. Serial dilutions were made of the testedsample in bacterial growth media. The test organisms were added to theproduct dilutions and incubated for growth. The dilutions of the testedsample that demonstrated no visible growth of the test organism wereplated to confirm lethality of the product. This procedure is a standardsusceptibility assay for antimicrobials and incorporates the intent ofthe American Society for Mocrobiology (ASM) methodology. Neutralizationwas confirmed at 70%.

Tested Sample:

The tested sample used was taken from a 250 ml Stock solution containing0.1 mg silver nitrate per 1 ml buffered solution. Each tested samplecontained the following components:

Glycerol: 10% v/v

Menthol: 0.05% w/v

TWEEN 20: 0.5% v/v

AgNO₃: 0.01% w/v

Tris: 50 mM

Test Organisms:

The following microbial strains were used in this study:

E. Coli ATCC No. 8739; Staphylococcus epidermidis ATCC No. 12228;Klebsiella pneumoniae ATCC No. 4352; and Staphylococcus aureus ATCC No.6538

Study Protocol:

Acceptance criteria: All positive controls should demonstrate growth ofthe target organism. All media and negative controls should demonstrateno growth of the target organism.

Culture Preparation: Tubes of soybean casein digest broth (SCDB) wereinoculated with stock cultures of bacteria and incubated at 37±2° C. for18-48 hours. Where necessary, culture concentrations were adjusted bydilution in 0.9% sodium nitrate (NaNO₃) to approximately 10⁸ colonyforming units (CFU)/ml using visual turbidity. On the day of testing, astandard plate count was performed on the suspension through dilution in0.9% NaNO₃ and plating in triplicate on neutralizer agar (NUAG) todetermine the starting titer.

MIC test procedure: The tested sample described hereinabove was two-foldserially diluted in sterile purified water (PURW). Next, 5 ml of eachdilution was added to 5 ml of 2× media. The final test dilutions rangedfrom 1:2 to 1:4096.

Two positive control tubes, per organism, were prepared by mixing 5 mlPURW with 5 ml of 2× media. Two negative control tubes, per sample, wereprepared by mixing 5 ml of the lowest sample dilution with 5 ml of 2×media. Two media control tubes were prepared by mixing 5 ml PURW with 5ml of 2× media. No test culture was added to either negative control ormedia control tubes.

All test sample dilution and positive control tubes were inoculated with0.05 ml of the test organism. All tubes were incubated at 37±2° C. for16-20 hours. Based upon growth, each tube dilution was scored as eitherpositive (+) or negative (0).

MBC test procedure: Dilutions demonstrating no growth were tested forMBC. A 0.1 ml aliquot was removed from each tube demonstrating nogrowth. Each dilution was plated in triplicate on NUAG. For a negativecontrol, sterile 2× media were plated onto NUAG. Positive Controls weremade by plating ≦100 CFU of the test organisms on NUAG. The test plateswere incubated at 37±2° C. for 2-4 days.

Neutralization Verification:

The lowest dilution of the tested sample tested that inhibited growth ofthe tests organism (MIC) was tested for neutralization recovery of thetest organism in 2×media. 0.1 ml aliquots of the tested sample dilutionwere plated in triplicate on NUAG. Three additional plates were preparedfor each organism as a titer control. Plates were spiked with ≦100 CFUof the test organism. The plates were incubated at 37±2° C. for 2-4days. The counts obtained from the titer control were compared to thoseof the test samples.

Results:

The results for the MIC and MBC are presented in Table 3.

Table 4 presents the neutralization results.

Testing met the acceptance criteria stated above.

TABLE 3 Test organism MIC Titer MBC Escherichia coli 1:8 1.7 × 10⁸CFU/ml 1:4 ATCC No. 8739 Staphylococcus 1:8 5.7 × 10⁷ CFU/ml NDepidermidis ATCC No. 12228 Klebsiella pneumoniae 1:8 1.3 × 10⁸ CFU/ml1:4 ATCC No. 4352 Staphylococcus aureus 1:4 1.4 × 10⁸ CFU/ml ND ATCC No.6538

TABLE 4 Percent Neutralization Sample Dilution Organism IdentificationRecovery 1:2 Escherichia coli 83 ATCC No. 8739 1:2 Staphylococcus 117epidermidis ATCC No. 12228 1:2 Klebsiella pneumoniae 117 ATCC No. 43521:2 Staphylococcus aureus 100 ATCC No. 6538

These results provide further support for the versatile efficaciousantimicrobial activity of the compositions described herein.

Example 4 Determination of USP Microbial Limit

Studies were conducted in order to determine the presence ofStaphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa,Salmonella sp. and other related organisms that may be objectionable orconsidered pathogenic in a non-sterile sample. The presence of theseorganisms indicates an environment that allows growth of similarpathogenic bacteria.

Study Protocol:

Acceptance Criteria: Raw plate count results should be within 30-300colony forming units (CFU) per plate or reported as an estimate.Aspergillus niger and other similar organisms can accurately be readwith only 8-80 CFU per plate. If no colonies are found, the results arereported as less than the sample dilution. The plate count is valid whenthe negative monitors are within the parameters established in thecurrent procedure. Positive controls for qualification shoulddemonstrate characteristic growth. Negative test monitors for theselective screening should not demonstrate growth of the indicatororganism.

Sample Preparation: The tested sample was prepared by combining 10 ml ofthe sample stock solution as described in Example 3 hereinabove with 90ml of 50 mM Tris-buffer pH 7.45. Ten-ml aliquots of this solution werethen placed in 90 ml of fluid casein digest-soy lecithin-polysorbate 20medium (FCDM) and acumedia lactose broth (ALBR).

Plate Counts: Using the pour plate technique, 1 ml of the sample wasplated in soybean casein digest agar (SCDA) in triplicate for bacterialcounts. The same procedure was performed using potato dextrose agar(PDXA) in triplicate for fungal counts. The SCDA plates were incubatedfor 48-72 hours at 30-35° C. while the PDXA plates were incubated for5-7 days at 20-25° C.

Sample Enrichment The samples were diluted in FCDM for Staphylococcusaureus and Pseudomonas aeruginosa screening, and ALBR for Salmonella andEscherichia coli screening.

The enrichment broths were allowed to incubate for 24-48 hours at 30-35°C.

Microbial Screening Following enrichment incubation, the broths weretransferred or streaked to the appropriate media for incubation asfollows:

Salmonella: Selenite-cystine broth and Tetrathionate broth-12-24 hoursat 30-35° C.; Brilliant green, Bismuth sulfite, and XLD agars-24-48hours at 30-35° C.;

P. aeruginosa: Cetrimide agar-24-48 hours at 30-35° C.;

S. aureus: Mannitol salt agar-24-48 hours at 30-35° C.;

E. coli: MacConkey agar-24-48 hours at 30-35° C.

Any suspect colonies were verified using biochemical tests.

Qualification:

Qualification testing was performed on the initial test of this sampletype as follows:

Twenty-four hour broth cultures of the four test organisms were grown at30-35° C. and diluted 1:1000. The organism aliquot used for inoculationwas ≦1% of the sample preparation. The inoculum was added within onehour of the dilution of the sample in the broths. The screeningprocedure was followed. All four test organisms must be recovered,demonstrating neutralization of the sample.

Results:

The tested solution passed qualification at the 1:10 dilution. Valuesobserved for both the total aerobic microbial count and the combinedmold and yeasts count were lower than 10, indicating “non detection”.

Routine analysis may therefore be performed at the 1:10 sample dilution.

In the pathogenic screening, results showed that all tested organismswere absent from the total aerobic and fungal counts. The plate countresults are not qualified for bacterial or fungal recovery.

Testing met the acceptance criteria.

Example 5 Anti-Microbial Susceptibility Test

Studies were conducted for screening a test sample containing silverions and menthol for antimicrobial activity. The challenge organismswere Staphylococcus aureus ATCC No. 6538, and Escherichia coli ATCC No.8739. The test procedure was an adaptation of the disk diffusion(Kirby-Bauer) method for antibiotic susceptibility testing.

Study Protocol:

Culture Preparation: Mueller-Hinton broth was inoculated with S. aureusand E. coli from stock cultures and incubated for 18-24 hours at 30-35°C. The test organisms were standardized using physiological saline toachieve a cell density equivalent to a McFatland Standard of 0.5. Theinoculum was used within 15 minutes after standardization.

Tested Sample:

The tested sample used was taken from a 250 ml Stock solution containing0.1 mg silver nitrate per 1 ml buffered solution. Each tested samplecontained the following components:

Glycerol: 10% v/v

Menthol: 0.05% w/v

TWEEN 20: 0.5% v/v

AgNO₃: 0.01% w/v

Tris: 50 mM

Test Performance: A sterile cotton swab was dipped into the standardizedinoculum, rotated several times, and pressed firmly on the inside wallof the tube above the fluid level to remove excess inoculum from theswab. The swab was streaked over the entire surface of theMueller-Hinton agar plate three times, with the plate rotatedapproximately 60° each time, then a final sweep was made around the agarrim. The lid was left agar for no longer than 15 minutes to allow anyexcess surface moisture to be absorbed. Sterile disks were placed on theagar plates using a pair of sterile forceps. One disk was placed in thecenter of each plate, pressing firmly so that the sample stayed in placeand contacted the agar surface evenly. Each disk was inoculated withapproximately 0.1 ml of the tested sample. For the negative controls,each disk was inoculated with sterile purified water.

The plates were incubated at 30-35° C. for approximately 24 hours. Thesamples were thereafter transferred to freshly prepared plates andincubated for approximately 24 hours. This procedure was repeated untilplates showed no zone of inhibition. The diameters of the zones ofinhibition (if present) were measured using calibrated caliperssensitive to 0.01 mm. The complete zone of inhibition, including thediameter of the sample, was measured.

Results:

The results are presented in Table 5, and demonstrate the susceptibilityof the bacteria to the tested sample.

TABLE 5 Diameter of the zone Diameter of the zone including sample (mm)including sample (mm) Test Organism 24 hours 48 hours Staphylococcus 116.24 No Zone aureus ATCC No. 2 16.34 No Zone 6538 3 15.33 No ZoneEscherichia coli 1 14.14 No Zone ATCC No. 8739 2 16.28 No Zone 3 12.53No Zone

Example 6 Comparative Study of Fungicidal/Fungistatic Activities of aSolution Containing Silver Ions and Menthol and of the Commercial B.Braun's PRONTOSAN

The antifungal activity of the composition described herein (a testedsample as described in Example 5 hereinabove) was tested and compared tothat of the commercially available products PRONTOSAN® (by B. Braun),MICROCYN® (by Occulus) and ANASEPT® (by Anacapa).

In Vitro Screening and Comparative Test of Fungicidal Activity:

SDB agar (100 μl) in ELISA plate well was inoculated with the testedstrain, and was subjected to initial growth by incubation for 24 hours,at 30° C.

100 μl of an aqueous solution of the tested substance (either thesolution containing silver ions and menthol, as described herein, or asolution of PRONTOSAN®, MICROCYN® or ANASEPT®, or of a 0.9% saline (ascontrol), were applied once, plates were incubated for 30 minutes at 30°C., the solutions were discarded, and 100 μl of SDB growth medium wasthereafter added, and plates were incubated for 30 minutes at 30° C. andthen medium was discarded. Plates were then incubated at 30° C. for 24days, and were examined and photographed daily.

FIG. 1 presents an image generally illustrating the appearance of afungal culture following 24 days incubation after 30 minutes exposure toa tested solution and subsequent washing: there is either no growth(Fungicidal effect, denoted as “F”), minimal poor growth (Inhibitoryeffect, denoted as “IN”) or full growth (No Effect, denoted “NE”).

FIGS. 2-4 present images illustrating the appearance of the exposedfungal culture, following 3, 8, 11 and 16 days incubation after 30minutes exposure to each of the tested solutions and subsequent washing.Tested solutions included: a composition containing silver ions andmenthol as described in Example 5 hereinabove (denoted “S”), PRONTOSAN®(denoted “P”), alternating subsequent exposures to a compositioncontaining silver ions and menthol (S) and PRONTOSAN® (P), (denoted“S+P” and “P+S”), MICROCYN® (denoted “M”) and ANACEPT® (denoted “A”) andtheir dilutions (1:2; 1:4; 1:8). Tested fungal strains were a clinicalstrain of Trichophyton rubrum (FIG. 2); Trichophyton rubrum NCPF 118commercial strain (FIG. 3); and a clinical strain of Microsporum canis(FIG. 4).

The data presented in FIGS. 2-4 clearly demonstrate the fungicidaleffect of a single treatment with the composition containing silver ionsand menthol, according to embodiments of the invention. The silverions-menthol composition exhibited an activity similar to that ofPRONTOSAN®; was active also at 1:2-1:4 dilutions; and was superior tothe hypochlorite-containing agents MICROCYN® and ANACEPT®. Furthermore,it was clearly demonstrated that subsequent treatments of the silverions-menthol composition and PRONTOSAN® provided a substantiallyimproved antifungal activity, with clear superiority of the order of thesilver ions-menthol composition first and PRONTOSAN® next, whichcombination demonstrated significant high antifungal efficacy even at1:8 dilutions on Trichophyton rubrum clinical strain (see, FIG. 2).

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. An antiseptic composition comprising, as activeingredients, menthol and a source of silver ions, and a pharmaceuticallyacceptable carrier, said source of silver ions generates silver ions insaid composition at a concentration of up to 6 mM.
 2. The antisepticcomposition of claim 1, wherein said menthol and said silver ions act insynergy.
 3. The antiseptic composition of claim 1, wherein aconcentration of said menthol ranges from 0.3 mM to 32 mM.
 4. Theantiseptic composition of claim 3, wherein said concentration of saidmenthol ranges from 0.6 mM to 6.4 mM.
 5. The antiseptic composition ofclaim 1, wherein a concentration of said silver ions ranges from 0.05 mMto 6 mM.
 6. The antiseptic composition of claim 1, wherein aconcentration of said silver ions ranges from 0.25 mM to 0.6 mM.
 7. Theantiseptic composition of claim 1, wherein a concentration of saidsilver ions ranges from 0.25 mM to 0.6 mM, and a concentration of saidmenthol ranges from 0.6 mM to 6.4 mM.
 8. The antiseptic composition ofclaim 1, wherein a concentration of said silver ions is 0.29 mM, and aconcentration of said menthol is 6.4 mM.
 9. The antiseptic compositionof claim 1, wherein a concentration of said silver ions is 0.29 mM, anda concentration of said menthol is 3.2 mM.
 10. The antisepticcomposition of claim 1, wherein a concentration of said silver ions is0.29 mM, and a concentration of said menthol is 0.64 mM.
 11. Theantiseptic composition of claim 1, wherein a concentration of saidsilver ions is 0.44 mM, and a concentration of said menthol is 3.2 mM.12. The antiseptic composition of claim 1, wherein a concentration ofsaid silver ions is 0.6 mM, and a concentration of said menthol is 3.2mM.
 13. The antiseptic composition of claim 1, wherein a concentrationof said silver ions is 0.6 mM, and a concentration of said menthol is0.64 mM.
 14. The antiseptic composition of claim 1, wherein said sourceof silver ions is selected from the group consisting of silver nitrate,silver sulfadiazine, an aminoalcohol-silver ion complex, an aminoacid-silver ion complex and a polymer-silver ion complex.
 15. Theantiseptic composition of claim 14, wherein said polymer-silver ioncomplex is polyvinylpyrrolidone (PVP)-silver ion complex.
 16. Theantiseptic composition of claim 1, further comprising a hyperosmoticagent.
 17. The antiseptic composition of claim 16, wherein saidhyperosmotic agent is selected from the group consisting of glycerol,polyethylene glycol, sodium chloride, a polysaccharide, mannitol,ammonium chloride, magnesium sulfate, and a combination thereof.
 18. Theantiseptic composition of claim 16, wherein said hyperosmotic agent isglycerol.
 19. The antiseptic composition of claim 18, wherein aconcentration of said glycerol ranges from 3% v/v to 15% v/v, based onthe total volume of said antiseptic composition.
 20. The antisepticcomposition of claim 16, wherein said hyperosmotic agent is polyethyleneglycol (PEG).
 21. The antiseptic composition of claim 20, wherein aconcentration of said PEG ranges from 8% v/v to 16% v/v based on thetotal volume of said antiseptic composition.
 22. The antisepticcomposition of claim 1, further comprising a solubilizing agent.
 23. Theantiseptic composition of claim 22, wherein said solubilizing agent isTWEEN
 20. 24. The antiseptic composition of claim 1, wherein saidpharmaceutically acceptable carrier is an aqueous solution.
 25. Theantiseptic composition of claim 1, being formulated as a topical dosageform.
 26. The antiseptic composition of claim 25, wherein said topicaldosage form is selected from the group consisting of a cream, a spray, agauze, a wipe, a sponge, non-woven fabrics, a cotton fabrics, a foam, asolution, a lotion, an ointment, a paste and a gel.
 27. An antiseptickit comprising a packaging material and the antiseptic composition ofclaim 1 being packaged in said packaging material.
 28. The antiseptickit of claim 27, being identified in print, in or on said packagingmaterial, for use in disinfecting a surface.
 29. The antiseptic kit ofclaim 28, wherein said surface is a bodily surface.
 30. The antiseptickit of claim 27, being identified in print, in or on said packagingmaterial, for use in the treatment of a wound.
 31. The antiseptic kit ofclaim 30, wherein said wound is selected from the group consisting of anacute wound, a chronic wound, a burn and a surgical wound.
 32. Theantiseptic kit of claim 31, wherein said chronic wound is selected fromthe group consisting of a diabetic ulcer, a venous ulcer and a pressureulcer.
 33. A method of disinfecting a surface, the method comprisingapplying an effective amount of the antiseptic composition of claim 1onto the surface, thereby disinfecting the surface.
 34. The method ofclaim 33, wherein said surface is a bodily surface, the method being fordisinfecting said bodily surface of a subject in need thereof.
 35. Themethod of claim 34, comprising topically applying said antisepticcomposition onto said bodily surface.
 36. The method of claim 34,wherein said bodily surface is a skin tissue.
 37. The method of claim34, being for treating an infection in said bodily surface.
 38. A methodof treating a wound in a subject in need thereof, the method comprisingapplying an effective amount of the antiseptic composition of claim 1 tothe wound area, thereby treating the wound.
 39. The method of claim 38,wherein the wound is selected from the group consisting of an acutewound, a chronic wound, a burn and a surgical wound.
 40. The method ofclaim 38, comprising topically applying said antiseptic composition ontosaid wound area.
 41. The method of claim 40, wherein topically applyingsaid antiseptic composition is performed by streaming a flow of theantiseptic composition over and through said wound area.
 42. A processof preparing the antiseptic composition of claim 1, the processcomprising admixing said source of silver ions, said menthol and saidpharmaceutically acceptable carrier, thereby obtaining the antisepticcomposition.
 43. The process of claim 42, further comprising admixing ahyperosmotic agent with the composition.
 44. The process of claim 42,further comprising admixing a solubilizing agent with the composition.45. The process of claim 44, wherein said solubilizing agent is admixedwith the menthol, prior to admixing the menthol with said source ofsilver ions and said carrier.
 46. The process of claim 44, wherein saidsolubilizing agent is TWEEN
 20. 47. A method of reducing a concentrationof silver ions in an antiseptic composition which comprises silver ions,the method comprising admixing with a source of the silver ions asynergistically effective amount of menthol, thereby reducing theconcentration of the silver ions in the antiseptic composition.
 48. Themethod of claim 47, wherein the concentration of the silver ions isreduced by at least 2-folds.
 49. The method of claim 47, wherein theconcentration of the silver ions is reduced by at least 10-folds.
 50. Amethod of increasing an antiseptic activity of an antiseptic compositionwhich comprises a source of silver ions at a concentration lower than 6mM, the method comprising admixing with the antiseptic composition asynergistically effective amount of menthol.